Skip to main content

Endogenous stem cell mobilization and localized immunosuppression synergistically ameliorate DSS-induced Colitis in mice

Abstract

Background

Stem cell therapy is a promising alternative for inflammatory diseasesĀ and tissue injury treatment. Exogenous delivery of mesenchymal stem cells is associated with instant blood-mediated inflammatory reactions, mechanical stress during administration, and replicative senescence or change in phenotype during long-term culture in vitro. In this study, we aimed to mobilize endogenous hematopoietic stem cells (HSCs) using AMD-3100 and provide local immune suppression using FK506, an immunosuppressive drug, for the treatment of inflammatory bowel diseases.

Methods

Reactive oxygen species (ROS)-responsive FK506-loaded thioketal microspheres were prepared by emulsification solvent-evaporation method. Thioketal vehicle based FK506 microspheres and AMD3100 were co-administered into male C57BL6/J mice with dextran sulfate sodium (DSS)Ā induced colitis. The effect of FK506-loaded thioketal microspheres in colitis mice were evaluated using disease severity index, myeloperoxidase activity, histology, flow cytometry, and gene expression by qRT-PCR.

Results

The delivery of AMD-3100 enhanced mobilization of HSCs from the bone marrow into the inflamed colon of mice. Furthermore, targeted oral delivery of FK506 in an inflamed colon inhibited the immune activation in the colon. In the DSS-induced colitis mouse model, the combination ofĀ AMD-3100 and FK506-loaded thioketal microspheres ameliorated the disease, decreased immune cell infiltration and activation, and improved body weight, colon length, and epithelial healing process.

Conclusion

This study shows that the significant increase in the percentage of mobilizedĀ hematopoietic stem cells in the combination therapy of AMD and oral FK506 microspheres may contribute to a synergistic therapeutic effect. Thus, low-dose local delivery ofĀ FK506 combined with AMD3100 could be a promising alternative treatment for inflammatory bowel diseases.

Background

Ulcerative colitis (UC) is a common gastrointestinal condition characterized by mucosal injury affecting the large intestines [1]. The treatment of UC involves 5-aminosalicylic acid, antibiotics, corticosteroids, and anti-TNF-Ī± or anti-CD3 antibody therapy [2]. However, long-term use of these drugs results in neurotoxicity, nephrotoxicity, and opportunistic infections. To minimize the adverse effects of drugs, local or targeted drug delivery systems to the colon have been introduced [3,4,5]. Physiological clues such as mucus secretion, variation in pH in different segments of the gastrointestinal tract, and a series of microbiome secreting enzymes have been used for targeted drug delivery [6]. However, loss of mucus-secreting goblet cells, altered pH, mangled microbiome, and altered GI transit time during UC affect the effectiveness of colon-targeted drug delivery systems where mucoadhesive polymer, bacterial enzyme-based prodrug, extend-release polymer, and pH-responsive materials have been applied to assist targeted-release of drugs to the colon. In that regard, disease-associated changes could be beneficial for targeted drug delivery to the colon. When reactive oxidative species (ROS)-responsive polymer was used to deliver the payload into the colon, accumulation of the payload in the colon significantly increased during UC [7]. SinceĀ only few proportion of the patients respond to the conventional treatment methods [8], alternative strategiesĀ for the effective treatment of inflammatory bowel disease (IBD) have been investigated.

MesenchymalĀ stem cell therapy is an alternative for IBD treatment due to its tissue regenerative and immunomodulatory effects [9]. However, the use of exogenously delivered mesenchymal stem cells (MSCs) is associated with the progression of replicative senescence, increased oxidative stress, loss of extracellular matrix, instant blood-mediated inflammatory reactions, and stress during injection [10, 11]. This compromises the therapeutic outcomes of the exogenous MSC-based therapy. Therefore, new approaches to mobilize the endogenous stem cells at the injury site toĀ accelerate tissue repairment and replacement are required. AMD3100 (AMD) is a Food and Drug Administration (FDA)-approved drug for the mobilization of hematopoietic stem cells (HSCs) from the bone marrow to the inflammation site [12, 13]. Several studies have used stem cell mobilization to enhance tissue repairment, transplantation acceptance, and treatment of inflammatory diseases [14,15,16]. As T and B cells exhibit C-X-C chemokine receptor type 4 (CXCR4) receptors, leucocyte mobilization alsoĀ occurs during stem cell mobilization [17]. Furthermore, it might aggravate the inflammation and immune reactions at the injured sites [18, 19]. Thus, the combination of AMD with FK506 not only enhances the stem cell mobilization ability of AMD [20] but also inhibits the inflammation by increasing regulatory immune cell population [21]. Many studies found that AMD therapy in combination with a low-dose immunosuppressive regimen effectively controls inflammation and immune activation [20, 21].

In our previous study, we evaluated the efficacy of FK506-loaded thioketal microspheres (TKMs) in the treatment of UC [7]. In the present study, we aimed to combine locally-delivered FK506 with CXCR4 antagonists to enhance epithelial regeneration by mobilizing endogenous stem cells from bone marrow and inhibiting immune cell infiltration. This study revealed, for the first time, that a significant increase in the percentage of hematopoietic stem cells in the combination therapy of AMD and oral FK506-TKM has a synergistic therapeutic effect. In addition, we have shown that a low dose of FK506 combined with AMD treats inflammatory bowel diseasesĀ in murine model.

Materials and methods

Preparation and characterization of ROS-responsive microspheres

The thioketal polymer was prepared and characterized as described previously [7]. Then, the emulsification solvent-evaporation method was used toĀ prepare ROS-responsive thioketal microspheres (TKMs) [7]. The freeze-dried TKMs were used for physical characterizations and in vivo applications. Morphological characterization was performed using the scanning electron microscope (SEM, S-4100, Hitachi, Tokyo, Japan), and drug loading capacity of FK506-loaded TKMs (FK506-TKMs) was determined by the High-performance liquid chromatography (HPLC) (Thermo Fisher Inc, Waltham, MA, USA) according to a previous method [22].

The microspheres were treated with 1Ā mM KO2 and 1Ā mM H2O2 for 48Ā h to confirm their ROS-responsive behavior. Next, degradation of FK506-TKMs was evaluated using the SEM, and in vitro release of FK506 from FK506-TKMs was performed in phosphate-buffered saline (PBS) (pH 7.4, 0.1% tween 20) with or without 1Ā mM H2O2 and 1Ā mM KO2. FK506 in the release sample was estimated using the HPLC.

Therapeutic effect of FK506-TKM and AMD3100 in colitis animal model

Eight to ten-week-old male C57BL6/J mice were purchased from Samtako (Seoul, Republic of Korea) and were used in the study according to the animal conduct of Yeungnam University, Republic of Korea (IACUC: YL 2018ā€“028) in compliance with ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines 2.0. After acclimatization for 1Ā week, mice were randomly divided into five groups (nā€‰=ā€‰7); Control, PBS, AMD, FK506-TKMs, and AMD-FK506-TKMs. Afterwards, colitis was induced by administering 3% w/v dextran-sulfate sodium (DSS, NY, USA) in drinking water for 7Ā days. The mice received daily gavage of PBS (nā€‰=ā€‰7), or FK506-TKMs (nā€‰=ā€‰7) (1Ā mg/kg/day). The group receiving AMD3100 (AMD, Selleck Chemicals, Houston, TX, USA) received AMD at the dose of 1Ā mg/kg via subcutaneous injection at 0, 2, and 4Ā days. Bodyweight decrease, stool consistency, and bleeding events were recorded throughout the study period. Furthermore, the disease severity index (DSI) was calculated as described previously [23]. Each animal was assessed for diarrhea, bleeding, and body weight loss, with scores ranging from 0 to 4 indicating severity. These scores were averaged to calculate the DSI for each animal. The animals were humanely euthanized using CO2 inhalation according to institutional guidelines and American Veterinary Association (AVMA) guidelines. After sacrificing mice on day 10 of DSS administration, colon length was measured.

Myeloperoxidase (MPO) assay

To determine the MPO assay, we applied the colorimetric method using O-dianisidine as a substrate, as described [7, 24]. Briefly, the colon was homogenized in 0.5% w/v hexadecyltrimethylammonium bromide (Sigma-Aldrich, St Louis, MO, USA) and prepared in 50Ā mM PBS (pH 6). The supernatant was used to observe the absorbance at 460Ā nm, where a change in absorbance was recorded for 15Ā min using the SPARK 10Ā M (TECAN, Untersbergstrasse, Grodig, Austria).

Hematoxylin & Eosin (H&E) staining

After feces removal from the isolated colon with PBS, the colon was fixed in 4% paraformaldehyde for approximately 24Ā h. Next, the fixed colon was soaked into 30% w/v sucrose for 24ā€’48Ā h, and thin sections were prepared using microtome. Then the tissue sections were rehydrated using a series of concentrations of alcohol, stained with hematoxylin and eosin, and finally dehydrated with ethanol gradient and covered with a coverslip using a mounting solution (Biomed, Foster City, CA. USA). An optical microscope (Nikon Eclipse Ti) was used to capture the images. The pathophysiology scoring based on histology was determined by the extent of infiltration of inflammatory cells, epithelial lining destruction, loss of goblet cells, hyperplasia, and cryptitis of the colon.

Immune cell isolation

Isolation of lymphocytes was performed from colonic lamina propria following the method described previously [25]. Briefly, the colon was washed with PBS to remove feces completely. Furthermore, to obtain single cells, colons were minced and subjected to collagenaseĀ digestion to obtain single cells. After digestion, RPMI with 10% FBS was used to neutralize the enzyme action and centrifuged to get cell pellets. The cell pellets were suspended in RPMI and were filtered using a 40Ā Ī¼m cell strainer. The collected cells were washed with PBS and stained with APC-conjugated anti-CD3 antibody for flow cytometry analysis. Similarly, colon-draining lymph nodes (c-MLN) were isolated following a previously described method [26]. For the determination of Th1 and Th17 differentiation, the lymphocytes obtained from c-MLN were stimulated in vitro with 750Ā ng/mL ionomycin (Calbiochem), GolgiStopā„¢, and 50Ā ng/mL phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich) for 4Ā h and the analysis was done through flow cytometry as described previously [7].

Evaluation of stem cell migration in the colon

To verify the migration of stem cells to the colon from bone marrow, the percentage of hematopoietic stem cells derived from bone marrow were evaluated. The washed colon was chopped, and collagenase digestion of the colonic tissue was performed using collagenase (1Ā mg/mL) and DNAse (10Ā Ī¼g/mL) for 30Ā min at 37Ā Ā°C with continuous stirring. Thus, obtained single cells were stained with Sca-1 and CD34 antibodies and the percentage double positive population of Sca-1+ and CD34+ stem cells was evaluated using FACS.

Western blotting

Colon tissues were lysed using RIPA-containing halt protease inhibitor cocktail (Thermo Scientific, 78,430). Proteins were quantified using the BCA assay kit and the same amount of proteins was resolved to 12% sodium dodecyl sulfateā€“polyacrylamide gel electrophoresis (SDS-PAGE), then transferred to an Immobilon-P transfer membrane (Millipore Corporation, Billerica, MA, USA). Then, 5% of skim milk was used for blocking the membrane and separately incubated with primary antibodies againstĀ COX-2 (1:1000; Cell Signaling Technology, 12282S), and Ī²-actin (1:1000; Cell Signaling Technology, 4970S) overnight at 4Ā Ā°C. Further, membranes were incubated with HRP-conjugated secondary antibodies for 1Ā h at room temperature and images were developed using a chemiluminescence detection kit (Thermo Scientific). The Ī²-actin level was used as a loading control. Expressed protein density was analyzed using GELQuantNET software (BiochemLab Solution, San Francisco, CA, USA).

Total RNA isolation and quantitative real-time polymerase chain reaction (qRT-PCR)

To conduct qRT-PCR, total RNA from colonic tissue was isolated using the triazol (Ambion Life Technology, Foster City, CA) and quantified by the NanoDrop technique using the SPARK 10Ā M (TECAN, Untersbergstrasse, Grodig, Austria). Then, cDNA was constructed using the GoScriptā„¢ reverse transcription system (Promega, Madison, WI). RT-PCR was conducted using the SYBER green to check the mRNA expression of TNF-Ī± and IFN-Ī³ in the colon samples. The primer sequences used in this study are listed in TableĀ 1.

TableĀ 1 Primer sequences

Statistical analysis

Statistical analysis was performed using the GraphPad Prism 5 software (GraphPad Software, Inc., La Jolla, CA, USA). Statistical values were calculated using a one-way analysis of variance or unpaired t-tests. Differences with p-values of less than 0.05 were considered statistically significant.

Results

Preparation and characterization of FK506-loaded microspheres

FK506-TKMs showed a uniform and spherical shape under SEM examination. FK506-TKMs showed ROS-dependent degradation when incubated with 1Ā mM H2O2 and KO2 for 48Ā h (Fig.Ā 1a). Moreover, in vitro release study in the presence or absence of 1Ā mM H2O2 and KO2 suggested that ROS-triggered release of FK506 from FK506-TKMs. The presence of ROS (1Ā mM H2O2 and KO2) in the release media accelerated the release of the FK506 from FK506-TKMs (Fig.Ā 1b). This suggests that the ROS-responsive behavior of FK506-TKMs provided on-demand availability of the payload at the site of inflammation (region with higher levels of ROS) compared to that of the normal tissues. Higher accumulation of drugs in the disease site ensures effectiveness while decreasing the symptoms of systemic adverse effects.

Fig.Ā 1
figure 1

Characterization of TKMs. a SEM images of FK506-TKMs without and with ROS treatment (1Ā mM of H2O2 and 1Ā mM of KO2) for 48Ā h. b Release profile of FK506. For the release study, 0.2% Tween 20 in PBS (pH: 7.5) with or without 1Ā mM of H2O2 and 1Ā mM of KO2 was used as release media

Effective alleviation of DSS-induced murine colitis by a combination of FK506-TKMs and AMD

FigureĀ 2a shows the schematic representation of the study design. The mice with DSS treatment showed marked loss of body weight. Treatment of AMD or FK506 alone did not prevent weight loss; however, the combination of AMD and FK506-TKMs significantly inhibited weight loss in the mice (Fig.Ā 2b). DSS treatment led to shortening of colon and this was recovered to a maximal extent in the combination group which further supports a synergistic effect of AMD and FK506-TKMs (Fig.Ā 2c, d). FigureĀ 2e shows disease severity index, which was calculated by considering body weight, presence of blood in stool, rectal bleeding, and stool consistency. This value was minimal in the AMDā€‰+ā€‰FK506-TKMs group (pā€‰<ā€‰0.001 vs PBS group). Neutrophil infiltration in colon was increased with DSS treatment. Maximum inhibition of neutrophil infiltration was observed in mice which received a combination therapy of AMD and FK506-TKMs (Fig.Ā 2f). Furthermore, under H&E examination, we observed a maximal damage of colonic epithelium when mice were treated with DSS only. Although AMD and FK506 alone treatments slightly improved the colon morphology, this damage was markedly attenuated when the mice were administered with AMD and FK506-TKMs (Fig.Ā 2g).

Fig.Ā 2
figure 2

Synergistic effects of AMD and FK506-TKMs on UC treatment. a Schematic representation of study timeline. b Body weight change of mice in control, PBS, AMD, FK506-TKMs, and AMDā€‰+ā€‰FK506-TKMs treated groups. The values represent meanā€‰Ā±ā€‰SEM (nā€‰=ā€‰7). c Representative image of the colon. d Quantitative estimation of colon length. The data represented the meanā€‰Ā±ā€‰SEM (nā€‰=ā€‰6) e Disease severity index. The data represents meanā€‰Ā±ā€‰SEM (nā€‰=ā€‰7). f MPO activity in the colon. The data is represented as meanā€‰Ā±ā€‰SEM (nā€‰=ā€‰7). g H&E staining of the colon. The mean score of H&E staining was calculated based on the extent of immune cell infiltration, goblet cell loss, and epithelial damage (nā€‰=ā€‰5). *pā€‰<ā€‰0.05, **pā€‰<ā€‰0.01, and ***pā€‰<ā€‰0.001. (Colon lengths of individual mice are presented in supplementary Fig.Ā 1)

Inhibition of immune cell infiltration and inflammation in colon by combination therapy of FK506-TKMs and AMD

To investigate the effect of the combinatorial approach of stem cell mobilization and local immunosuppression on immune cell infiltration in the colon, we estimated the percentage of CD3+ cells in colonic lamina propria. DSS-treatment significantly increased infiltration of CD3+ cells in the colon of PBS treated group (pā€‰<ā€‰0.001) compared to the control. However, the treatment with subcutaneous delivery of AMD, oral delivery of FK506-TKMs, and a combination of AMDā€‰+ā€‰FK506-TKMs (AMD: pā€‰<ā€‰0.01 vs. PBS group; FK506-TKMs: pā€‰<ā€‰0.01 vs. PBS group; AMDā€‰+ā€‰FK506-TKMs: pā€‰<ā€‰0.001 vs. PBS group) significantly inhibited the infiltration of CD3 cells compared to PBS-treated group (Fig.Ā 3a, b). Moreover, to evaluate the inflammation in the colon, inflammatory proteins and genes were evaluated in the colon. The PBS-treated group showed markedly higher expression of COX-2, suggesting the aggravation of inflammation in the colon. The treatment of AMD, FK506-TKMs, and AMDā€‰+ā€‰FK506-TKMs attenuate the expression of COX-2 where the AMDā€‰+ā€‰FK506-TKMs treated group showed the lowest expression of COX-2 (Fig.Ā 4a). This suggested the effectiveness of combinational therapy in inhibiting colonic inflammation. Moreover, a significant decrease in TNF-Ī± and IFN-Ī³ was observed in FK506-TKMs (pā€‰<ā€‰0.05 vs. PBS group) and AMDā€‰+ā€‰FK506-TKMs (pā€‰<ā€‰0.01 vs. PBS group) compared to PBS treated group (Fig.Ā 4b, c).

Fig.Ā 3
figure 3

Inhibition of immune cell infiltration in the colon. a Representative image of flow cytometric analysis representing the percentage of CD3+ cells in control, PBS, AMD, FK506-TKMs, and AMDā€‰+ā€‰FK506-TKMs treated groups. b Percentage of CD3+ T cells in the colon. The data represents meanā€‰Ā±ā€‰SEM (nā€‰=ā€‰5). **pā€‰<ā€‰0.01, and ***pā€‰<ā€‰0.001

Fig.Ā 4
figure 4

Inhibition of inflammatory protein and genes expression in the colon. a Protein expression of COX-2 in the colon b mRNA expression of TNF-Ī± in the colon of PBS, AMD, FK506-TKMs, and AMDā€‰+ā€‰FK506-TKMs treated groups. c mRNA expression of IFN-Ī³ in the colon of PBS, AMD, FK506-TKMs, and AMDā€‰+ā€‰FK506-TKMs treated groups. The data represents meanā€‰Ā±ā€‰SEM (nā€‰=ā€‰4). *pā€‰<ā€‰0.05, **pā€‰<ā€‰0.01. (Full western blot data are presented in supplementary Fig.Ā 2)

Inhibition of Th1 and Th17 differentiation of CD4+ T cells

The inflammation in the colon is followed by the activation of the T cells triggering the differentiation of naĆÆve T cells to Th1 or Th17 type pro-inflammatory cells in the colon draining mesenteric lymph nodes. To evaluate the effect on Th1/Th17 differentiation in lymphocytes, ionomycin/PMA re-stimulated lymphocytes were evaluated for INF-Ī³ and IL-17 secreting cells in CD4 cells. In the PBS treated group, higher activation of CD4 cells to INF-Ī³ secreting Th1 cells was observed, which was significantly decreased in the TKMs and AMDā€‰+ā€‰TKMs group (Fig.Ā 5a, b).

Fig.Ā 5
figure 5

Inhibition of Th1/Th17 differentiation of CD4 T cells. a The percentage of IFN+CD4+ T cells in cMLN determined by FACS. b The percentage of IL 17+CD4+ T cells in cMLN determined by FACS. Data representsā€‰Ā±ā€‰SEM (nā€‰=ā€‰4). *pā€‰<ā€‰0.05, **pā€‰<ā€‰0.01, and ***pā€‰<ā€‰0.001

Stem cell migration from bone marrow to colon

Stem cells have an immunomodulatory effect of suppressing inflammation and the regenerative effect for tissue remodeling and repairment. However, stem cells migrate towards the injury by sensing the cytokines produced at the infected sites. This study evaluated the effect of mobilization of endogenous stem cells combined with immunosuppressive drugs in the infected colon. In PBS and TKMs treated groups, higher hematopoietic stem cells (HSCs) (Sca-1+CD34+ cells) migrated from bone marrow than untreated control mice. However, in AMD and AMDā€‰+ā€‰TKMs treated groups, there was a significantly higher accumulation of HSCs compared to the PBS treated group (Fig.Ā 6a, b). This suggests that AMD effectively mobilizes the HSCs from bone marrow to the inflamed colon.

Fig.Ā 6
figure 6

AMD enhanced the stem cell migration to colon. a Representative FACS figure showing the percentage of CD34+Sca-1+ b Percentage of these stem cells migrated from bone marrow to colon. Data representsā€‰Ā±ā€‰SEM (nā€‰=ā€‰4). *pā€‰<ā€‰0.05 and ***pā€‰<ā€‰0.001

Discussion

Stem cell therapy is a promising alternative for treating a variety of diseases. However, the exogenously transferred stem cells undergo apoptosis and senescence; hence, affecting MSC-based therapy. Therefore, there is a need to mobilize endogenous stem cells from bone marrow. This study used a combination of stem cells mobilizer and low-dose FK506 using ROS-responsive particles. From the results, we confirmed that theĀ local delivery of FK506 and stem cell mobilization led to a significant improvement in the therapeutic outcomes in UC. In our previous study, the local delivery of FK506 using newly synthesized ROS responsive polymer improved the therapeutic effect compared to free-FK506. Despite the inhibition of immune cell infiltration into the colon, reduction of body weight during colitis was not prevented with the use of the FK506 microspheres alone. Therefore, additional interventions are needed to prevent bodyweight reduction. To prevent immune cell activation and maintain normal body weight, we used the FK506-TKMs in combination with the CXCR4 antagonist.

AMD (Plerixafor or Mozobil) is a CXCR4 antagonist and is an FDA-approved drug for stem cell mobilization from bone marrow to peripheral blood. CXCR4 is a chemokine receptor highly expressed in HSC. CXCR4 signaling is involved in the retention of HSC in the bone marrow. In contrast, the blockage of CXCR4 resulted in the mobilization of HSC from bone marrow to the peripheral bloodstream. Due to the activity of AMD in mobilizing HSCs from bone marrow to the infected sites, it has been applied for the treatment of several tissue injuries. For example, single-dose AMD was effective in functional recovery of myocardial infractions through neovascularization [27], and the topical application of AMD resulted in the recovery of wounds in diabetic mice [28].

Furthermore, the mobilized HSCs inhibit inflammation through the high expression of costimulatory PD-L1 resulting in inhibition of immune cells [29]. On the other hand, enhancement of angiogenesis and vasculogenesis was associated with the tissue repairing effect of AMD [27, 28]. In colitis, AMD modulated the colonic claudin expressions and improved intestinal barrier function [30].

Mature lymphocytes, monocytes, and neutrophils are affected by CXCR4 signaling. Their migration, homing, and retention is influenced by the chemokine receptors, including CXCR4. Several researchers have reported that the mobilization of the immune cells from bone marrow to the blood and lymph organs or spleen also occurs by using AMD [31]. The effect of AMD was enhanced in several studies when used in combination with low-dose immunosuppressive drugs [20, 29, 32,33,34]. The use of FK506 in combination with AMD enhances the mobilization of stem cells through increasing SDF-1 production and assists in the migration of stem cells to the inflammation sites [20]. The low-dose FK506 pulls the stem cells from bone marrow niches where SDF-1 (induced by FK506) acts as a strong pulling agent for the circulating stem cells to the injured tissue resulting in an increased accumulation of stem cells at the injured tissue promoting rapid repairment. In our study, we used a very low dose of FK506 to target the site of inflammation using ROS responsive microspheres. This prevents systemic toxicity of FK506 while exerting a potent immunosuppressive activity locally at the inflamed colon. Therefore, in the combination treatment group, the overall stem cell mobilization was found to be similar to the AMD only group in our study. The combination of AMD and targeted FK506 delivery enhanced therapeutics effect in colitis compared to FK506-TKMs or AMD only by increasing stem cell recruitment into the colon and decreasing the immune recruitment or activation locally at the inflammation site.

Conclusion

This study reported a synergistic drug therapy to alleviate DSS-induced experimental colitis effectively. Locally delivered calcineurin inhibitor and HSC mobilizing agent prevented infiltration of immune cells and colitis progression in DSS-fed mice. Furthermore, FK506-TKMs showed improved effects in colitis compared to free FK506 through local immunosuppression. Furthermore, the immobilized HSCs led to the migration of stem cells into the colon and the acceleration of tissue regeneration through immunomodulation and epithelial regeneration. Therefore, the combined therapeutic regimen proposed might be a milestone in treating colitis.

Availability of data and materials

Not applicable.

Abbreviations

UC:

Ulcerative colitis

Anti-TNF-Ī±:

Anti-tumor necrosis factor- Ī±

GI tract:

Gastrointestinal tract

ROS:

Reactive oxygenĀ species

IBD:

Inflammatory bowel disease

MSCs:

Mesenchymal stem cells

FDA:

Food and drug administration

HSCs:

Hematopoietic stem cells

CXCR4:

C-X-C chemokine receptor type 4

TKMs:

Thioketal microspheres

AMD:

AMD-3100

HPLC:

High performance liquid chromatography

SEM:

Scanning electron microscope

DSS:

Dextran-sulfate sodium

MPO:

Myeloperoxidase

H & E:

Hematoxylin and Eosin

c-MLN:

Colon-drainingĀ mesenteric lymph nodes

PMA:

Phorbol 12-myristate 13-acetate

FACS:

Fluorescence-activated cell sorting

SDS-PAGE:

Sodium dodecyl sulfateā€“polyacrylamide gel electrophroresis

COX-2:

Cyclooxygenase-2

HRP:

Horseradish peroxidase

IFN-Ī³:

Interferon- Ī³

SDF-1:

Stromal cell-derived factor 1

References

  1. Head KA, Jurenka JS. Inflammatory bowel disease part I: ulcerative colitisā€“pathophysiology and conventional and alternative treatment options. Altern Med Rev. 2003;8(3):247ā€“84.

    PubMedĀ  Google ScholarĀ 

  2. Tamboli CP. Current medical therapy for chronic inflammatory bowel diseases. Surg Clin North Am. 2007;87(3):697ā€“725.

    ArticleĀ  PubMedĀ  Google ScholarĀ 

  3. Sinha V, Kumria R. Colonic drug delivery: prodrug approach. Pharm Res. 2001;18(5):557ā€“64.

    ArticleĀ  CASĀ  PubMedĀ  Google ScholarĀ 

  4. Wei H, et al. Selective drug delivery to the colon using pectin-coated pellets. PDA J Pharm Sci Technol. 2008;62(4):264ā€“72.

    CASĀ  Google ScholarĀ 

  5. Ahmed IS, Ayres JW. Comparison of in vitro and in vivo performance of a colonic delivery system. Int J Pharm. 2011;409(1ā€“2):169ā€“77.

    ArticleĀ  CASĀ  PubMedĀ  Google ScholarĀ 

  6. Zhang S, Langer R, Traverso G. Nanoparticulate drug delivery systems targeting inflammation for treatment of inflammatory bowel disease. Nano Today. 2017;16:82ā€“96.

    ArticleĀ  CASĀ  PubMedĀ  PubMed CentralĀ  Google ScholarĀ 

  7. Regmi S, et al. Inflammation-triggered local drug release ameliorates colitis by inhibiting dendritic cell migration and Th1/Th17 differentiation. J Control Release. 2019;316:138ā€“49.

    ArticleĀ  CASĀ  PubMedĀ  Google ScholarĀ 

  8. Lightner AL. Stem cell therapy for inflammatory bowel disease. Clin Transl Gastroenterol. 2017;8(3):e82.

    ArticleĀ  PubMedĀ  PubMed CentralĀ  Google ScholarĀ 

  9. Mehler VJ, Burns C, Moore ML. Concise review: exploring immunomodulatory features of mesenchymal stromal cells in humanized mouse models. Stem Cells. 2019;37(3):298ā€“305.

    ArticleĀ  PubMedĀ  Google ScholarĀ 

  10. Lukomska B, et al. Challenges and controversies in human mesenchymal stem cell therapy. Stem Cells Int. 2019;2019:10.

    ArticleĀ  Google ScholarĀ 

  11. Baldari S, et al. Challenges and strategies for improving the regenerative effects of mesenchymal stromal cell-based therapies. Int J Mol Sci. 2017;18(10):2087.

    ArticleĀ  PubMedĀ  PubMed CentralĀ  Google ScholarĀ 

  12. De Clercq E. MozobilĀ®(Plerixafor, AMD3100), 10 years after its approval by the US food and drug administration. Antiviral Chem Chemother. 2019;27:2040206619829382.

    ArticleĀ  Google ScholarĀ 

  13. Liles WC, et al. Mobilization of hematopoietic progenitor cells in healthy volunteers by AMD3100, a CXCR4 antagonist. Blood. 2003;102(8):2728ā€“30.

    ArticleĀ  CASĀ  PubMedĀ  Google ScholarĀ 

  14. Chen L, et al. Combination of G-CSF and AMD3100 improves the anti-inflammatory effect of mesenchymal stem cells on inducing M2 polarization of macrophages through NF-ĪŗB-IL1RA signaling pathway. Front Pharmacol. 2019;10:579.

    ArticleĀ  CASĀ  PubMedĀ  PubMed CentralĀ  Google ScholarĀ 

  15. Toupadakis CA, et al. Mobilization of endogenous stem cell populations enhances fracture healing in a murine femoral fracture model. Cytotherapy. 2013;15(9):1136ā€“47.

    ArticleĀ  PubMedĀ  PubMed CentralĀ  Google ScholarĀ 

  16. Hu X, et al. Chimeric allografts induced by short-term treatment with stem cell-mobilizing agents result in long-term kidney transplant survival without immunosuppression: a study in rats. Am J Transplant. 2016;16(7):2055ā€“65.

    ArticleĀ  CASĀ  PubMedĀ  PubMed CentralĀ  Google ScholarĀ 

  17. Kean LS, et al. Significant mobilization of both conventional and regulatory T cells with AMD3100. Blood. 2011;118(25):6580ā€“90.

    ArticleĀ  CASĀ  PubMedĀ  PubMed CentralĀ  Google ScholarĀ 

  18. Vogel M, et al. A limited role for AMD3100 induced stem cell mobilization for modulation of thoracic trauma outcome. Shock. 2022;57(6):260ā€“7.

    ArticleĀ  CASĀ  PubMedĀ  Google ScholarĀ 

  19. Watanabe M, et al. Dual effect of AMD3100, a CXCR4 antagonist, on bleomycin-induced lung inflammation. J Immunol. 2007;178(9):5888ā€“98.

    ArticleĀ  CASĀ  PubMedĀ  Google ScholarĀ 

  20. Lin Q, et al. Pharmacological mobilization of endogenous stem cells significantly promotes skin regeneration after full-thickness excision: the synergistic activity of AMD3100 and tacrolimus. J Investig Dermatol. 2014;134(9):2458ā€“68.

    ArticleĀ  CASĀ  PubMedĀ  Google ScholarĀ 

  21. Okabayashi T, et al. Mobilization of host stem cells enables long-term liver transplant acceptance in a strongly rejecting rat strain combination. Am J Transplant. 2011;11(10):2046ā€“56.

    ArticleĀ  CASĀ  PubMedĀ  PubMed CentralĀ  Google ScholarĀ 

  22. Pathak S, et al. Preparation of high-payload, prolonged-release biodegradable poly(lactic-co-glycolic acid)-based tacrolimus microspheres using the single-jet electrospray method. Chem Pharm Bull (Tokyo). 2016;64(2):171ā€“8.

    ArticleĀ  CASĀ  PubMedĀ  Google ScholarĀ 

  23. Wirtz S, et al. Chemically induced mouse models of intestinal inflammation. Nat Protoc. 2007;2:541.

    ArticleĀ  CASĀ  PubMedĀ  Google ScholarĀ 

  24. Regmi S, et al. Heterospheroid formation improves therapeutic efficacy of mesenchymal stem cells in murine colitis through immunomodulation and epithelial regeneration. Biomaterials. 2021;271:120752.

    ArticleĀ  CASĀ  PubMedĀ  Google ScholarĀ 

  25. Acharya S, et al. Amelioration of experimental autoimmune encephalomyelitis and DSS induced colitis by NTG-A-009 through the inhibition of Th1 and Th17 cells differentiation. Sci Rep. 2018;8(1):7799.

    ArticleĀ  PubMedĀ  PubMed CentralĀ  Google ScholarĀ 

  26. Houston SA, et al. The lymph nodes draining the small intestine and colon are anatomically separate and immunologically distinct. Mucosal Immunol. 2016;9(2):468ā€“78.

    ArticleĀ  CASĀ  PubMedĀ  Google ScholarĀ 

  27. Jujo K, et al. CXCR4 blockade augments bone marrow progenitor cell recruitment to the neovasculature and reduces mortality after myocardial infarction. Proc Natl Acad Sci. 2010;107(24):11008ā€“13.

    ArticleĀ  CASĀ  PubMedĀ  PubMed CentralĀ  Google ScholarĀ 

  28. Nishimura Y, et al. CXCR4 antagonist AMD3100 accelerates impaired wound healing in diabetic mice. J Investig Dermatol. 2012;132(3):711ā€“20.

    ArticleĀ  CASĀ  PubMedĀ  Google ScholarĀ 

  29. Fiorina P, et al. Targeting the CXCR4ā€“CXCL12 axis mobilizes autologous hematopoietic stem cells and prolongs islet allograft survival via programmed death ligand 1. J Immunol. 2011;186(1):121ā€“31.

    ArticleĀ  CASĀ  PubMedĀ  Google ScholarĀ 

  30. Xia X-M, et al. CXCR4 antagonist AMD3100 modulates Claudin expression and intestinal barrier function in experimental colitis. PLoS ONE. 2011;6(11):e27282.

    ArticleĀ  CASĀ  PubMedĀ  PubMed CentralĀ  Google ScholarĀ 

  31. Bernardini G, et al. CCL3 and CXCL12 regulate trafficking of mouse bone marrow NK cell subsets. Blood. 2008;111(7):3626ā€“34.

    ArticleĀ  CASĀ  PubMedĀ  Google ScholarĀ 

  32. Zhai R, et al. Pharmacological mobilization of endogenous bone marrow stem cells promotes liver regeneration after extensive liver resection in rats. Sci Rep. 2018;8(1):3587.

    ArticleĀ  PubMedĀ  PubMed CentralĀ  Google ScholarĀ 

  33. Cameron AM, et al. Chimeric allografts induced by short-term treatment with stem cell mobilizing agents result in long-term kidney transplant survival without immunosuppression: ii, study in miniature swine. Am J Transplant. 2016;16(7):2066ā€“76.

    ArticleĀ  CASĀ  PubMedĀ  Google ScholarĀ 

  34. Dā€™Addio F, et al. Autologous nonmyeloablative hematopoietic stem cell transplantation in new-onset type 1 diabetes: a multicenter analysis. Diabetes. 2014;63(9):3041ā€“6.

    ArticleĀ  PubMedĀ  Google ScholarĀ 

Download references

Acknowledgements

The authors declare that they have not used artificial intelligence (AI)-generated work in this manuscript.

Funding

This research was supported by the National Research Foundation of Korea (NRF) funded by the Ministry of Science and ICT (Grant Nos. 2021K1A3A1A20002609 and RS-2023ā€“00272815), by the Bio & Medical Technology Development Program of the NRF funded by the Korean government (MSIT) (Grant No. 2022M3A9G8017220), and by a Korean Fund for Regenerative Medicine (KFRM) grant funded by the Ministry of Science and ICT and the Ministry of Health & Welfare (Grant Nos. 22A0205L1 and 23A0205L1).

Author information

Authors and Affiliations

Authors

Contributions

SR and SP contributed to conceptualization, investigation, validation, formal analysis, and writing-original draft; DC, JOK, JWN, HSK, HLJ, and DR participated in writing-reviewing and editing the manuscript; JHS, SY, and JHJ contributed to supervision, writing-reviewing, and editing the manuscript. All authors read and approved the final manuscript.

Corresponding authors

Correspondence to Jong-Hyuk Sung, Simmyung Yook or Jee-Heon Jeong.

Ethics declarations

Ethics approval and consent to participate

The animal study protocol was approved by Institutional Animal Care and Use Committee (IACUC) of Yeungnam University, Republic of Korea, entitled ā€œCombined effect of hematopoietic stem cell immobilization and local immune suppression for treating ulcerative colitisā€. This study was approved on February 15, 2018, with approval number IACUC: YL 2018-028. All animal experiments were conducted under university guidelines.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Regmi, S., Pathak, S., Chaudhary, D. et al. Endogenous stem cell mobilization and localized immunosuppression synergistically ameliorate DSS-induced Colitis in mice. Stem Cell Res Ther 15, 167 (2024). https://doi.org/10.1186/s13287-024-03777-2

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1186/s13287-024-03777-2

Keywords