The Loss of YTHDC1 in Gut Macrophages Exacerbates Inflammatory Bowel Disease

Abstract The nuclear N 6‐methyladenosine (m6A) reader YT521‐B homology‐domain‐containing protein 1 (YTHDC1) is required to maintain embryonic stem cell identity. However, little is known about its biological functions in intestinal‐resident macrophages and inflammatory bowel disease (IBD). Herein, it is demonstrated that macrophage‐specific depletion or insufficiency of YTHDC1 accelerates IBD development in animal models. On the molecular basis, YTHDC1 reduction in IBD‐derived macrophages is attributed to Zinc finger protein 36 (ZFP36)‐induced mRNA degradation. Importantly, transcriptome profiling and mechanistic assays unveil that YTHDC1 in macrophages regulates Ras homolog family member H (RHOH) to suppress inflammatory responses and fine‐tunes NME nucleoside diphosphate kinase 1 (NME1) to enhance the integrity of colonic epithelial barrier, respectively. Collectively, this study identifies YTHDC1 as an important factor for the resolution of inflammatory responses and restoration of colonic epithelial barrier in the setting of IBD.


Table S2
Sequences of primers involved in the study
Monocytes would differentiate into macrophages in 7 days.

Colitis models
Colitis models were established according to previous publications [3]. were provided 2.5% DSS (Thermo Fisher Scientific) dissolved in autoclaved tap water for 7 days followed by a 7-day tap water treatment based on published procedure [3]. Survival, body weights, rectal bleeding and stool consistency were monitored every day. Clinical and histological scores were evaluated as described before [3]. Scoring system for clinical index based on stool consistency, weight loss and the severity of intestinal bleeding. Il10 KO mice develop spontaneous colitis in 6-8 weeks after birth [4].

Bone marrow-derived macrophage (BMDM) and peritoneal macrophage (PM)
BMDMs were cultured and isolated following previous publications [5]. Briefly, bone marrow cells were harvested from the legs of mice by cold PBS, followed by red blood cells elimination. Cells were cultured with 30% L929 conditioned media for differentiation. BMDMs were ready to use after being cultured for 7 days. In other experiments, isolated bone marrow cells were infected with a lentivirus for 48 hours before differentiation.
For peritoneal macrophage isolation, mice were intraperitoneally injected with 5 ml cold PBS followed by PBS fluids collection. Cell suspension was centrifuged to pellet macrophages. Peritoneal macrophages were cultured in RPMI 1640 media with 10% heat-inactivated FBS and 1% P/S.

Histology and Immunostaining
Fresh colon tissues were harvested, cut longitudinally, flushed with cold PBS and fixed immediately in 4% formaldehyde overnight. Then, the colon tissues were were imaged under a microscope.

Bone marrow transplantation
Bone marrow transplantation was performed according to published procedure [6]. Briefly, recipient mice were subject to γ-irradiation of 1050 rads (200 rads/min) to deplete original bone marrow cells. 6 hours after irradiation, the recipient mice were transplanted with bone marrow cells (5x10 6 cells/mouse) from donor mice through retra-orbital injection. Two months later, the transplanted mice were ready for colitis induction.

Fecal microbiota transplantation (FMT)
FMT was performed based on a published paper [7]. Briefly, donor feces from IBD patients and healthy individuals were harvested and dissolved in anaerobic PBS   Table S2.
After 24 hours, BMDMs were harvested, and luciferase activities were assessed using a Luciferase Assay System kit (Promega) following the manufacture's protocols.
pRL-TK renilla luciferase reporter vectors were transfected to serve as internal controls.

CRISPR/Cas9-modulated gene knockout.
sgRNA sequences targeting the Ythdf1 gene were inserted into lentiCRISPRv2 vector (Addgene) by the BsmBI restriction enzyme. Lenti-vector and packaging plasmids were transfected into HEK293T cells together. After 48 hours, packaged lentivirus in culture medium of HEK293T cells was harvested and transduced into macrophages supplemented with polybrenes (4 µg/ml). Details on sgRNA were provided in Table S2.

Macrophage depletion and reconstitution.
Macrophages in mice were depleted using clodronate-containing liposomes following previous publications [5,8]. In brief, mice were intravenously administered 5 mg/ml clodronate-liposomes (0.2 ml/mouse) from Encapsula NanoSciences. 48 hours later, mice were reconstituted with fresh 2x10 6 BMDMs suspended evenly in PBS with or without lentivirus infection via intravenous injection. 24 hours after reconstitution, these mice were used for the induction of colitis using TNBS.

Colonic epithelial cells, lamina propria cells, macrophages, T cells, B cells, dendritic cells and neutrophils isolation
Fresh mice colon tissues were harvested, cut longitudinally, flushed with cold PBS and cut into 3 mm fragments. Freshly harvested human colon biopsies were cut into 3 mm fragments on ice directly. Colonic epithelial cells were isolated as described before [9]. In brief, the fragmented tissues were rotated in cold PBS including EDTA (10 mM) for 30 min in cold room. After centrifugation and multiple washes with cold PBS, tissues were pipetted up and down to separate epithelial layer and lamina propria, followed by crypts enrichment through a cell strainer (70-μm).
The enriched epithelial cells were then purified by 20%/40% Percoll. Colonic lamina propria cells were enriched as described [10]. Briefly, the fragmented tissues were Neutrophils from human or mouse blood were isolated by human neutrophil isolation kit (Miltenyi Biotec) or mouse neutrophil isolation kit (Miltenyi Biotec), respectively.

Flow cytometry and cell sorting
Flow cytometry analysis was carried out according to previous publications [11].

RNA affinity chromatography
RNA affinity chromatography experiments were implemented as previously described [12]. In brief, ssRNA oligonucleotides possessing adenosine or m 6

Western blot
Cells or tissues were lysed in RIPA buffer (Invitrogen) with protease inhibitor cocktail (Roche

RT-qPCR and m 6 A-IP-qPCR
Total RNAs from cells or tissues were isolated by TRIzol reagent (Invitrogen), followed by reverse transcription using a reagent kit (TOYOBO) to synthesize cDNA.
Quantitative PCR (qPCR) was carried out using a SYBR real-time PCR kit (TOYOBO). Relative gene levels were normalized to GAPDH expression. For  Table S2.

Cross-linking and RNA Immunoprecipitation-qPCR (CLIP-qPCR)
CLIP assays were performed according to previous publications [8] with slight modifications. Briefly, macrophages were stimulated with 4-thiouridine (100 mM) in 37°C for 14 hours, followed by cross-linking with UV irradiation at 0.15 J/cm 2 on ice.
Cells were lysed in lysis buffer and then harvested for immunoprecipitation. Before immunoprecipitation, RNase T1 (0.1 U/µl) was added for 10-min incubation at 22°C, and 10% of lysates were saved as input controls, while the other 90% of samples were mixed with antibody-protein G magnetic bead complex for 1-hour rotation in cold room. Immunoprecipitated RNAs were extracted with TRIzol reagents and quantified by qPCR.

Assessment of transcription rate
Transcription rate experiments were implemented following a published paper [13]. Briefly, 5-Ethynyl Uridine (EU, 0.5 mM) was added to macrophages for 0-min, 20-min, 40-min or 60-min treatments separately. Total RNAs were isolated by TRIzol reagent and nascent RNAs were purified by the Click-iT Nascent RNA Capture Kit (Invitrogen) according to the manufacturer's instructions. RNA amounts were evaluated by RT-qPCR.

mRNA decay assay
Macrophages were added with actinomycin D (5 mg/ml) for 0-hour, 2-hour, 4-hour or 8-hour treatments prior to total RNAs extraction. mRNA amounts of each time point were detected by RT-qPCR. The mRNA level of 0 hour was used for normalization.

Cell viability and proliferation assays
Cell viability was measured using the CellTiter-Glo 2.0 kit (Promega) following the standard protocols. Cell proliferation at different time points was detected by the Cell Proliferation Assay kit (Sigma) according to the manufacturer's instructions. The initial number of cells for each group is 5000 at day 0.

NF-κB activity assay.
NF-κB activity of macrophages was measured using NF-κB luciferase reporter kit (BPS Bioscience). Macrophages with or without LPS/IFNγ stimulations were co-transfected with NF-κB luciferase reporter as well as pRL-TK renilla luciferase reporter. The Dual Luciferase Assay System was used to monitor luciferase activities.

Permeability Measurement
Colonic epithelial cells and macrophages were co-cultured in 0.4 µm Transwell chambers (Corning) for permeability assays until monolayers of epithelial cells were formed. Transepithelial electrical resistance (TER) of colonic epithelial cell monolayer was measured using an electrical resistance system (Millicell ERS-2 Voltohmmeter, Millipore). In another experiment, 200 µL of 10 mg/ml 4000 Da FITC-Dextran (Sigma) was added to the apical side of cell monolayer. 4 hours later, 100 µL aliquots of culture medium were collected from the basal chamber for measurements [14]. For in vivo studies, mice were fasted for 6 hours prior to being gavaged with 4000 Da FITC-dextran orally at 200 mg/kg. 2 hours later, blood was harvested from each mouse for detections [14]. Serum or medium FITC-dextran was monitored by a fluorescence spectrometer at 530 nm wavelength.

Cytokines detection
TNFα, IFNγ, IL-6, IL-17, IL-1β, IL-12p70, IL-23 and IgA concentrations of cells, tissues or cell culture media were measured by various ELISA kits (Biolegend) following the manufacturer's protocols. For cells and tissues, 100,000 cells or 1 mg tissues were lysed in 300 µl complete extraction buffer (150 mM NaCl, 100 mM Tris (pH 7.4), 1mM EDTA, 1 mM EGTA, 0.5% sodium deoxycholate, 1% Triton X-100 and protease inhibitor cocktail) and homogenized with a homogenizer on ice, followed by 2-hour rotation in cold room. Lysates were then centrifuged at 13,000 rpm in cold room for 20 min and supernatants were saved for further analysis.

Cell co-culture
Transwell inserts (Corning, Polycarbonate, 0.4 mm) were selected to establish the co-culture system. For HCT116 and macrophage co-culture, BMDMs were plated in the 24-well carrier plate at the density of 1x10 6 /well. HCT116 cells with or without siNME1 transfection were cultured into the insert at the density of 1x10 5 /well until monolayers were formed, followed by TER measurement and FITC-dextran permeability assay. In another experiment, HCT116 cells were cultured in the plate and BMDMs were plated into the insert for immunofluorescence staining. For macrophage and T cell co-culture, naive CD4 + T cells were enriched from the spleen of wild type mice using CD4 T cell isolation kit (Miltenyi Biotec) and cultured in the anti-CD3 and anti-CD28 coated plate. BMDMs were plated and treated with LPS/IFNγ for 8 hours, and then the supernatant of BMDMs was pipetted out and BMDMs were washed 3 times with cold PBS to remove extracellular LPS and cytokines. T cells were grown in the 24-well carrier plate at the density of 1x10 6 /well and macrophages were cultured into the insert at the density of 0.5x10 6 /well. After 3-day co-culture, T cells were isolated for analyses.

RNA-seq data analysis
The raw data of sequencing were processed by trim_galore v0.6.5, and then reads were mapped using STAR v2.6.1d with mm10 reference genome. R and edgeR package were used to calculate differential expression. P-values were analyzed for multiple testing by the false discovery rate (FDR) correction of Benjamini and Hochberg. R and package clusterProfiler were selected to perform gene ontology biological process enrichment analysis.

Statistics
Statistical analysis was conducted on normally distributed data sets.
Representative data are presented as the mean ± SD. Sample size (n) was provided in figure legends. GAPDH was used for the normalization of qPCR data and β-actin was used for western blot data normalization. The student's two-tailed t-test was used for the statistical significance of two group comparisons. A one way or two-way ANOVA was performed for the statistical significance of three or more group comparisons. The log-rank test was conducted for the statistical significance of animal survival rates. P < 0.05 was considered to be statistically significant. SPSS 17.0 software was used to perform statistical analyses.