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 to 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 1 mM KO2 and 1 mM H2O2 for 48 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 1 mM H2O2 and 1 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 2018–028) in compliance with ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines 2.0. After acclimatization for 1 week, mice were randomly divided into five groups (n = 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 7 days. The mice received daily gavage of PBS (n = 7), or FK506-TKMs (n = 7) (1 mg/kg/day). The group receiving AMD3100 (AMD, Selleck Chemicals, Houston, TX, USA) received AMD at the dose of 1 mg/kg via subcutaneous injection at 0, 2, and 4 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 50 mM PBS (pH 6). The supernatant was used to observe the absorbance at 460 nm, where a change in absorbance was recorded for 15 min using the SPARK 10 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 24 h. Next, the fixed colon was soaked into 30% w/v sucrose for 24‒48 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 collagenase 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 40 μm 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 750 ng/mL ionomycin (Calbiochem), GolgiStop™, and 50 ng/mL phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich) for 4 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 (1 mg/mL) and DNAse (10 μg/mL) for 30 min at 37 °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 sulfate–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 against COX-2 (1:1000; Cell Signaling Technology, 12282S), and β-actin (1:1000; Cell Signaling Technology, 4970S) overnight at 4 °C. Further, membranes were incubated with HRP-conjugated secondary antibodies for 1 h at room temperature and images were developed using a chemiluminescence detection kit (Thermo Scientific). The β-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 10 M (TECAN, Untersbergstrasse, Grodig, Austria). Then, cDNA was constructed using the GoScript™ reverse transcription system (Promega, Madison, WI). RT-PCR was conducted using the SYBER green to check the mRNA expression of TNF-α and IFN-γ in the colon samples. The primer sequences used in this study are listed in Table 1.

Table 1 Primer sequencesStatistical 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.