1.1. Mice
Seven weeks old C57BL/6 male mice were purchased from Harlan/Envigo and maintained at the Ruijin Hospital of Shanghai Jiaotong University School of Medicine animal facility under specific pathogen-free conditions. Animal care and experimentation were consistent with Laboratory animal Guideline for animal experiment report of China and were approved by the Institutional Animal Care and Use Committee at Shanghai jiaotong University. All methods are reported in accordance with ARRIVE guidelines for the reporting of animal experiments in the study.
1.2. DSS induced UC mouse model
Ten C57BL/6 male mice were randomly divided into 2 groups (control group: N; DSS group: UC) with 5 mice in each group. UC mice were given 3.5% DSS in drinking water for 4 days followed by water for 3 days. Control group was given water only. The mice were weighed daily and disease activity index (DAI, partial Mayo score) was recorded (Table 1 and Table2). Mice were sacrificed on day 8, at which point, the UC mice had a body weight reduction of 15%. The mouse colons were stained with hematoxylin and eosin (HE).
Table 1
Assessment of inflammation by disease activity index*
Score
|
Weight
|
0
|
the same
|
1
|
5–10% weight loss
|
2
|
10–15% weight loss
|
3
|
15–20% weight loss
|
4
|
20% weight loss
|
*: Amiri et al,.(2019). Complement Ther Med, 45:262–268. |
Table 2
Assessment of inflammation by disease activity index *.
Score
|
Stool
|
Bleeding
|
0
|
normal
|
no blood
|
2
|
loose stool
|
presence
|
4
|
diarrhea
|
gross blood
|
*: Amiri et al,.(2019). Complement Ther Med, 45:262–268. |
1.3. Tissue collection, colonoid generation and culture
Crypts were isolated from the mouse colon as previously described. 7 Briefly, colonic tissue was collected in sterile phosphate-buffered saline (PBS) and kept on ice throughout the entire manipulation. The tissue was cut longitudinally and feces were washed off with cold complete chelating solution (CCS). The tissue was minced to approximately 2 mm pieces using sterile scissors. The tissue fragments were transferred to a 15mL tube with cold CCS to wash and allowed them to settle by gravity. The supernatants were removed and the cold CCS were added into the 15mL tube to wash the fragments again. The process was performed three to five times until the supernatants were completely cleared The supernatants were removed and the fragments were incubated in 10mM EDTA in CCS for 1 hour at 4°C on an orbital shaker for shaking at 200 revolutions per minute (rpm). After 1 hour, the tissue fragments were collected in a new 15 mL tube and fetal bovine serum (FBS, Corning 35010CV) was added with the final concentration of 20% FBS, which allowed the released crypts to remain in the supernatant when the fragments settled to the bottom of the tube. The supernatant contained crypts which were collected and spun down at 1,200 rpm for 10 min at 4°C. The crypt pellet was resuspended in 30% Matrigel (Corning 356231) in 24-well plate and incubated at 37°C for 15 min to allow Matrigel to polymerize. 0.5 mL complete medium containing 10% FBS, 1% Pen/Strep (Quality Biological 120095721), 1%L-glutamine (Gibco 35050061), 1% sodium pyruvate (Gibco 11360), 2% non-essential amino acids (Gibco 11130), 2.5% 1 M HEPES (Quality Biological 118089721), 50µM 2-mercaptoethanol (Sigma M6250), 50 ng/ml recombinant murine EGF (Invitrogen PMG8041),100 ng/ml recombinant murine Noggin (Peprotech 250–38),500 ng/ml recombinant human R-spondin (R&D Systems4645-RS) were added to each well. The complete medium was replaced every two days.
1.4 In vitro lymphocyte culture
Splenic lymphocytes were isolated by Ficoll density gradient. They were then cultured in RPMI (Roswell Park Memorial Institute) media for 3 days, in 96-well plates pre-coated with 2µg/ml of anti-CD3ε (17A2) with1 µg/ml of soluble anti-CD28 (37.51).
1.5. Colonoid-lymphocyte co-culture
Colonoids were extracted from matrigel as described, 7,8 and washed with cold CMGF− [advanced F/12 DMEM) (Gibco 12634010), 1% Pen/Strep, 1%L-glutamine and 2.5% 1 M HEPES]. Lymphocytes were collected from 96-well plates, washed with RPMI 1640 with 10% FBS and 1% Pen/Strep, centrifuged at 1400 rpm and 4°C for 5 min and counted. Celltrace™ CFSE cell proliferation kit (Invitrogen, C34544) was used to stain lymphocytes and Celltrace™ far-red cell proliferation kit (Invitrogen, C34564) was used to dye the colonoids, according to per manufacturer protocol. Approximately 100,000 lymphocytes and 20 colonoids were then co-cultured in each well of a 24-well plate with 500 µl of complete culture medium with 30% matrigel. The culture medium was replaced every 2 days and the co-culture system was re-plated in fresh 30% matrigel twice a week. 9
1.6. Quantitative reverse transcription-polymerase chain reaction (qRT‐PCR)
To evaluate mRNA expression of interleukin (IL) 6, IL-17A, tumor necrosis factor alpha (TNF-α), IL-1β, and IL-12 from cells in the co-culture system, total RNAs was extracted from colonoid-lymphocyte co-culture system. qRT-PCR was performed on a Step One Plus real-time system (AB Applied Biosystems). GAPDH was used as an endogenous control. Relative levels of gene expression were quantified using the equation, −ΔΔ2Ct (Pan et al., 2020). Three replicates were run on the same plate for each sample. The mouse primers utilized are listed in Table 3. 10
Table 3
Primer sequences used for qRT-PCR
Gene
|
|
Sequence (5′–3′)
|
IL-6
|
Forward
|
5′- CAC TTC ACA AGT CGG ACG CT -3
|
Reverse
|
5′- TGC CAT TGC ACA ACT CTT TTC T -3′
|
IL-17A
|
Forward
|
5′- TCCAGAAGGCCCTCAGACTA − 3′
|
Reverse
|
5′- ACACCCACCAGCATCTTCTC-3′
|
TNF-α
|
Forward
|
5′- TACTGAACTTCGGGGTGATTGGTCC-3′
|
Reverse
|
5-CAGCCTTGTCCCTTGAAGAGAACC-3′
|
IL-12
|
Forward
|
5’-TTG AAC TGG CGT TGG AAG CAC G-3’
|
Reverse
|
5’-CCA CCT GTG AGT TCT TCA AAG GC-3’
|
IL-1b
|
Forward
|
5’-TGC CAC CTT TTG ACA GTG ATG-3’
|
Reverse
|
5’-ATG TGC TGC TGC GAG ATT TG-3’
|
GAPDH
|
Forward
|
5′- TTG ATG GCA ACA ATC TCC AC -3′
|
Reverse
|
5′- CGT CCC GTA GAC AAA ATG GT -3′
|
1.7. Inflammatory cytokine measurements
Three cytokines (IL-6, IL-17A, and TNF-α) were measured in the co-culture media by U-PLEX Biomarker Group multiplex assays (Meso Scale Diagnostic, Rockville, MD) following the manufacturer’s instructions. 11 IL-6, IL-17A, and TNF-α were reported as pg contained in the total volume of culture supernatant (20 ul).
1.8. Drug testing
Colonoid-lymphocyte co-cultures from DSS mice were plated in 96-well plates and cultured for 3 days. Next, the culture media was replaced to drug-containing media. Mesalamine, azathioprine and prednisone at a concentration of 10 µM, respectively, were used to treat co-cultures. Next, we chose other small molecules that had been previously identified in a computational study as effective in IBD (prochlorperazine, topiramate and vorinostat). 12 DMSO served as a control (used to dilute the drugs). The effects of drug treatment were evaluated in respect to the impact on certain cytokines in the supernatant (IL-6, IL-12, IL-17A, TNF-α, and IL-1β). These cytokines were measured at 72h after starting the drug treatment. Next, the co-cultured cells were collected and RNA extracted with the TRI zol method.
1.9. Statistics
Groups were compared using one-way ANOVA with Tukey’s multiple comparisons test and one-way ANOVA with Dunnett’s multiple comparisons test. 13 All tests were performed with GraphPad Prism version 5 software. Statistical significance for comparisons was assigned at *P < 0.05, **P < 0.01 and ***P < 0.001.