{"id":202412,"date":"2023-10-24T20:00:00","date_gmt":"2023-10-25T00:00:00","guid":{"rendered":"https:\/\/platohealth.ai\/organoids-transplantation-attenuates-intestinal-ischemia-reperfusion-injury-in-mice-through-l-malic-acid-mediated-m2-macrophage-polarization-nature-communications\/"},"modified":"2023-10-25T13:52:43","modified_gmt":"2023-10-25T17:52:43","slug":"organoids-transplantation-attenuates-intestinal-ischemia-reperfusion-injury-in-mice-through-l-malic-acid-mediated-m2-macrophage-polarization-nature-communications","status":"publish","type":"post","link":"https:\/\/platohealth.ai\/organoids-transplantation-attenuates-intestinal-ischemia-reperfusion-injury-in-mice-through-l-malic-acid-mediated-m2-macrophage-polarization-nature-communications\/","title":{"rendered":"Organoids transplantation attenuates intestinal ischemia\/reperfusion injury in mice through L-Malic acid-mediated M2 macrophage polarization – Nature Communications","gt_translate_keys":[{"key":"rendered","format":"text"}]},"content":{"rendered":"
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Mice with the same genotype were assigned randomly to each group by an independent experimenter, but no specific randomization method was employed. Additionally, no statistical methods were utilized to pre-estimate the sample size. Experimenters responsible for histological analyses, IF and relevant laboratory tests were blinded to the grouping of the mice.<\/p>\n

Animals<\/h3>\n

Male C57BL\/6\u2009J WT mice (6-8 weeks old) were provided by the Animal Center at the Nanfang Hospital of Southern Medical University (Guangzhou, China). Lgr5-EGFP-IRES-CreERT2 (Lgr5-GFP) mice were obtained from Jackson Laboratory (Ban Harbor, ME, USA). Breeding pairs of SOCS2 knocked-out mice were kindly provided by Zai-Long Chi (Wenzhou Medical University). SOCS2\u2013\/\u2013<\/sup> mice in the C57BL\/6 genetic background were generated by using the CRISPR\/Cas9-mediated genome engineering technology. The deletion of the exon 3 fragment was carried out using gRNA1 (TTG GCA GTC GTT TTT CTA GT CGG) and gRNA2 (ATT CAG CTA AAA CTA CCT AA GGG) generated by Cyagen Biosciences (Guangzhou, China). All animals were housed in an animal room under specific pathogen-free (SPF) conditions (12\u2009h light-dark cycle, temperature: 22\u2009\u00b1\u20091\u2009\u00b0C and humidity: 55\u2009\u00b1\u20095%) and had ad libitum access to standard mouse chow (MD17121, MEDICIENCE, Jiangsu, China) and water. The animals used for the experiment were assigned randomly to the different groups. All animal procedures described here were approved by the local Animal Care and Use Committee of the Nanfang Hospital of Southern Medical University (Guangzhou, China) and carried out in line with the National Institutes of Health guidelines. All animal handling were according to the policy of the China Animal Welfare guidelines and under the supervision of the ethics committee of Southern Medical University. Mice were gavaged with 0.2\u2009mL\/20\u2009g ddH2O or 250\u2009mg\/kg MA (M7397, Sigma-Aldrich, St. Louis, MO, USA) once a day consecutively for five days until surgery. The mice used here were randomized into the following experimental groups: (i) control; (ii) organoid transplantation; (iii) PBS liposome; (iv) clodronate liposome; (v) vehicle; and (vi) MA. Mice in the vehicle groups were gavaged 0.2\u2009mL\/20\u2009g of ddH2<\/sub>O once a day for five days and used as the vehicle. Approval of this study was obtained from the Nanfang Hospital animal ethics committee (approval number: IACUC-LAC-20220508-001).<\/p>\n

Establishment of the mouse intestinal I\/R injury model<\/h3>\n

Before surgery, all animals were deprived of food for 18\u2009h, but with ad libitum access to water. Mice were anesthetized using 2-3% inhalant isoflurane, and their skin was prepared before surgery. To establish the model, the small intestine was exposed through a mid-abdominal incision, and the superior mesenteric artery (SMA) was clipped for 50\u2009min using a non-invasive microvascular artery clamp, followed by reperfusion, as previously described4<\/a><\/sup>. In brief, successful ischemia was identified through the pale color of the small intestine mucosa, while valid reperfusion was determined based on the reappearance of pink color. For liquid resuscitation, 1\u2009mL of saline was immediately injected subcutaneously after the release of the clamp. After the abdominal incision was closed with suture, 1\u2009mg\/ml butorphanol (Jiangsu Hengrui Medicine Co., Ltd., Jiangsu, China) was injected subcutaneously for pain management. The survival of the mice was monitored every 6\u2009hours after releasing the artery clamp and continued to be monitored for a study period of 7 days.<\/p>\n

Culture of mouse intestinal organoids<\/h3>\n

Mice were sacrificed, the small intestine excised, immersed in sterile cold PBS, cut lengthwise, and rinsed using sterile cold PBS to wash away the intestinal contents. The intestine was then cut into pieces (2-4\u2009mm) and placed in a 50-mL centrifuge tube which contained sterile cold PBS (prepared in advance), and a 2-ml pipette was used to wash the pieces via pipetting up and down repeatedly 15-20 times with sterile cold PBS. Then pieces were incubated for 15\u2009min with 10\u2009mL of 30\u2009mM EDTA in PBS at room temperature. Subsequently, the reagent was removed, the intestinal sections were washed with PBS, and the supernatant fractions enriched in crypts were collected using a 70-\u00b5m cell strainer. These fractions were centrifuged at 400\u2009\u00d7\u2009g<\/i> for 5\u2009min, following which the supernatant was removed. The precipitate was resuspended in 5\u2009mL cold Dulbecco\u2019s Modified Eagle Medium\/Nutrient Mixture F-12 (DMEM\/F12) solution (Gibco, Thermo Fisher Scientific, Waltham, MA, USA), crypts were counted using an inverted microscope, and the required amount of liquid was aspirated into a centrifuge tube and centrifuged for 5\u2009min at 400 \u00d7 g. The precipitate was collected and resuspended in the same amount of organoid medium, including mouse epidermal growth factor (mEGF), Noggin, and R-spondin (ENR medium), and Matrigel (356231, BD Biosciences, Franklin Lakes, NJ, USA). The ENR medium contained DMEM\/F12 media (Gibco), Primocin (100\u2009\u00b5g\/mL, InvivoGen, San Diego, CA, USA), N2 supplement (1X, Gibco), B27 supplement (1X, Gibco), mouse epidermal growth factor (50\u2009ng\/mL, PeproTech, Cranbury, NJ, USA), R-Spondin 1 (500\u2009ng\/mL, R&D Systems, Minneapolis, MN, USA), and murine Noggin (100\u2009ng\/mL, R&D Systems). Next, the suspension (50\u2009\u00b5L) was quickly inoculated on preheated cell culture dishes to form a dome-shaped gelatinous structure. The dishes were then placed in a 37\u2009\u00b0C incubator for 20\u2009min. ENR medium was added to the culture dishes for organoid culture after the Matrigel was solidified.<\/p>\n

Orthotopic transplantation<\/h3>\n

On transplantation day, freshly isolated crypts and sorted small-intestinal Lgr5+<\/sup> stem cells were prepared as described in the methods. Mature mouse organoids were removed from the Matrigel and washed with cold DMEM\/F12 solution. The isolated organoids, freshly isolated crypts and sorted small-intestinal Lgr5+<\/sup> stem cells were then resuspended in Matrigel and DMEM\/F12 (1:4) and kept on ice for no more than 30\u2009min. Approximately 3\u00d7106<\/sup> sorted small intestinal Lgr5+<\/sup> stem cells, 8000 freshly isolated crypts, and 5000 organoids per 200\u2009\u03bcL of fluid were obtained. Control DMEM\/F12 and Matrigel were prepared under the same conditions but in the absence of organoids. Mice were anesthetized prior to induction of intestinal I\/R injury. Immediately after the clips were released, 200\u2009\u00b5L organoid solution was injected once into the duodenal lumen from the proximal to the distal portion of the small intestine using a 1-mL syringe with a 23-gauge needle and the control DMEM\/F12 and Matrigel without organoids were injected in the same manner. Subsequently, the small intestine was carefully returned to the abdominal cavity, the incision was closed, and the anus was attached using tissue glue. Six hours after surgery, the glue was removed.<\/p>\n

Blood sample and tissue processing<\/h3>\n

The mice were anesthetized before being sacrificed. Collection of blood samples was accomplished by removing the eyeballs, following which the blood samples were centrifuged for 15\u2009min at 400\u2009\u00d7\u2009g<\/i> to collect sera. After the small intestines were removed, intestinal tissue was fixed using paraformaldehyde (PFA) for histopathological analysis. The lumen was then flushed with cold PBS, and the remaining tissue was collected and frozen using liquid nitrogen. After the cecum was excised, its contents were carefully squeezed out and harvested using sterile forceps. All collected samples were stored at \u221280\u2009\u00b0C before use.<\/p>\n

Fluorescence<\/i> imaging<\/h3>\n

Prior to fluorescence detection, the entire small intestine was dissected from the mice. Fluorescence was detected using an Ami HTX Optical Imaging System (Spectral Instruments Imaging, Tucson, AZ, USA), and images were taken at 470\u2009nm excitation, 570\u2009nm emission, and 60\u2009s exposure time using small binning (resolution). All images were analyzed and processed using ANALYSIS ONLY Aura 4.0.7\u2009M.<\/p>\n

Organoid-derived conditioned medium<\/h3>\n

Mouse intestinal organoids were cultured in six-well plates expanded for five to seven days. Then the organoids were cultured in the medium refreshed by DMEM\/F12 in advance for 24\u2009h, and the medium was collected and filtered through a 0.22 \u03bcm filter (431219, CORNING, Corning, NY, USA) after the 24\u2009h culture to obtain the organoid-conditioned medium.<\/p>\n

Histology and immunohistochemistry<\/h3>\n

For frozen-embedded sections, 4% PFA (Sigma-Aldrich) was used for the fixation of tissue and organoid samples, and 15% and 30% sucrose solutions were used to sequentially dehydrate samples, which were then embedded, frozen in OCT (Sakura Finetek), and sectioned at 6\u2009\u03bcm intervals. For paraffin-embedded sections, 4% PFA was used to fix the samples, after that, samples were dehydrated using an ascending alcohol gradient, embedded in paraffin, and sectioned into 4 \u03bcm slices. Sample sections were then stained with hematoxylin and eosin (H & E). Evaluation of the degree of small intestinal injury was accomplished using Chiu\u2019s method with some modifications64<\/a><\/sup>. In brief, the modification pathological scoring criteria are as follows: 0, normal intestinal mucosa and intestinal villus. 1, formation of Gruenhagen cavity begins at the tip of the intestinal villus. 2, formation of Gruenhagen cavity and slight damage of glands. 3, formation and enlargement of subepithelial gaps, congested and engorged capillaries. 4, moderate detachment of epithelium from lamina propria and gland damage. 5, partial loss of villus at the tip. 6, obvious loss of villus and dilated capillaries. 7, loss of lamina propria and significant gland damage. 8, beginning of digestion and decomposition of the lamina propria. 9, hemorrhage and formation of ulcers. The depth of the crypt was measured from the bottom to the opening. Paraffin-embedded sections were pressure-cooked in Tris-EDTA (pH 9.0) for 10\u2009min before use. For permeabilization, cells and sections were incubated using 0.1% or 0.3% Triton X-100 (Thermo Fisher Scientific) in PBS at room temperature for 10\u2009min and then washed and blocked in 10% goat serum (Gibco) at room temperature for 1\u2009h. The primary and secondary antibodies used were diluted in 3% bovine serum albumin (BSA) in PBS. The primary antibodies that were used are as follows: anti-ZO-1 (1:400, ab216880, Abcam), anti-occludin (1:200, ab216327, Abcam), anti-Ki-67 (1:200, ab279653, Abcam), anti-OLFM4 (1:200, 39141\u2009S, Cell Signaling Technology), anti-Muc2 (1:100, 27675-1-AP, Proteintech), anti-lysozyme (1:500, A0099, Dako) and anti-mannose receptor (1:200, ab64693, Abcam); DAPI (1:500, D9542, Sigma-Aldrich) was used to stain the nuclei. The positive cells were quantified in five randomly chosen crypts per image. Images were captured using an ortho fluorescence microscope (Axio Imager D2, Carl Zeiss), analyzed, and processed via ImageJ software (National Institutes of Health, Bethesda, MD, USA).<\/p>\n

RNA extraction and quantitative real-time polymerase chain reaction (qRT-PCR)<\/h3>\n

TRIzol reagent (10296028; Thermo Fisher Scientific) was used to extract the total RNA from the tissues, organoids, and cells. RNA purity and concentration were measured using a spectrophotometer (Nano Drop OneC; Thermo Fisher Scientific). Reverse transcription of RNA to complementary DNA (cDNA) was performed using a cDNA reverse transcription kit (FSQ-101, TOYOBO). Real-time PCR was performed using an ABI Q6 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA), according to the SYBR Green detection protocol (QPK-201, TOYOBO). Finally, 18\u2009S ribosomal RNA was used as a housekeeping gene, and the 2-\u0394\u0394CT<\/sup> method was used to normalize data. Primers used here are listed (Supplementary Table 1<\/a>).<\/p>\n

Cytokine measurements<\/h3>\n

Cell culture medium and serum samples were thawed at room temperature. Next, the concentrations of IL-6, IL-1\u03b2, TNF-\u03b1, and IL-10 in the samples were measured using enzyme-linked immunosorbent assay (ELISA) kits (KE10002, KE10003, KE10007, and KE10008, Proteintech) in accordance with the manufacturer\u2019s instructions.<\/p>\n

Flow cytometry<\/h3>\n

The small intestine was washed using PBS, and Peyer\u2019s patches and fat tissue were removed47<\/a><\/sup>. The intestines were opened lengthwise, cut into pieces and incubated with 10\u2009mL, 5\u2009mM EDTA for 15\u2009min, and the supernatant containing epithelial cells was discarded. The remaining tissues were transferred into RPMI-1640 medium containing collagenase IV (0.5\u2009mg\/mL, C5138, Sigma-Aldrich), DNase I (100 U, 10104159001, Roche), and incubated at 37\u2009\u00b0C for 1\u2009h. Subsequently, the supernatants were discarded, the tissue fragments were washed repeatedly with PBS, and the supernatants containing laminar cells were collected. The fractions were resuspended in Percoll (GE17-0891-01, Sigma-Aldrich) after centrifugation, and the middle interface layer was harvested after density gradient centrifugation. The cell precipitate was resuspended for subsequent use. Small intestinal macrophages were cultured in 10% fetal bovine serum (FBS) and stained with APC-Cy7\u2013conjugated anti-CD45 (I3\/2.3, A15395, Thermo Fisher Scientific), BV510-conjugated anti-mouse CD11b antibody (clone M1\/70, 101263, Biolegend), FITC-conjugated mouse F4\/80 (clone BM8, 123108, Biolegend), and sorted.<\/p>\n

Spleen tissues were carefully pulverized using a 70-\u03bcm cell strainer, rinsed in PBS, collected in 15-mL centrifuge tubes, and centrifuged at 400 \u00d7 g for 5\u2009min. The precipitate was resuspended in erythrocyte lysis buffer (Miltenyi Biotec, Bergisch Gladbach, Germany) and incubated for 5\u2009min on ice. Subsequently, digestion was terminated using cold PBS and the sample was centrifuged at 400\u2009\u00d7\u2009g<\/i> for 5\u2009min. The remaining cell pellets were resuspended within cold PBS and stored at 4\u2009\u00b0C until further use.<\/p>\n

After rinsing the bone marrow tissues derived from bone marrow cavities with DMEM (Gibco), the suspensions were centrifuged. Red blood cells were lysed using lysis buffer and resuspended in cold PBS for later use.<\/p>\n

Blood samples were centrifuged at 400\u2009\u00d7\u2009g for 15\u2009min to obtain sera, and the remaining samples were diluted with 1\u2009mL PBS. The diluted blood samples were then transferred gently into 15-mL centrifuge tubes containing 1\u2009mL Ficoll Histopaque (P8900, Solarbio Life Sciences, Beijing, China). This led to the formation of two distinct layers, which were then centrifuged for 15\u2009min at density gradients. The cell precipitate was resuspended for testing.<\/p>\n

The cells were incubated with a protein transport inhibitor cocktail (554724, BD Biosciences) at 37\u2009\u00b0C for 4\u2009h and Fc block (553141, BD Pharmingen, Franklin Lakes, NJ, USA) at 4\u2009\u00b0C for 15\u2009min. The cells were then incubated at 4\u2009\u00b0C for 30\u2009min in the dark for surface staining with fluorophore-conjugated antibodies before being fixed, permeabilized, and incubated with intracellular cytokine staining antibodies against IL-10 (clone JES5-16E3) and CD206 (clone C068C2). Flow cytometry acquisition was performed using an LSRFortessa X-20 Multidimensional HD Flow Cytometer (BD Biosciences), while data analysis was performed using FlowJo software (Tree Star Inc., Ashland, Or, USA). The antibodies used are listed in Supplementary Table 2<\/a>.<\/p>\n

Small intestinal crypts were isolated from Lgr5-EGFP-IRES-CreERT2 mice, as described above. Crypts were further dissociated into single cells using TrypLE Express (Invitrogen, Waltham, MA, USA). Enhanced GFP (EGFP) stem cells were gated and sorted using a MoFlo XDP ultra-speed flow cell sorting system (Beckman Coulter, Brea, CA, USA).<\/p>\n

Depletion of intestinal macrophages<\/h3>\n

Two hundred microliters of clodronate- or PBS-loaded liposomes (CP-005-005, LIPOSOMA, Groningen, The Netherlands) were intravenously injected into mice thrice on alternating days prior to intestinal I\/R to deplete intestinal macrophages.<\/p>\n

Participants<\/h3>\n

Twenty-three participants undergoing coronary artery bypass graft or elective cardiac valve replacement surgery, at the cardiac surgery department of the Nanfang Hospital of Southern Medical University (Guangzhou, China), were consecutively recruited between September 2020 and November 2021. All participants were aged between 18 and 75 years. Exclusion criteria included those with chronic digestive system diseases, previous gastrointestinal surgery, chronic kidney disease, confirmed or suspected intestinal ischemia\/necrosis, and those who had used prebiotics, laxatives or antidiarrheals within one week, or antibiotics within three months before the start of the study. No significant differences between the demographic and health-related characteristics of the groups were observed. Written informed consent was obtained from all patients. Ethical committee approval of this study was obtained from the Nanfang Hospital of Southern Medical University (approval number: NFEC-202009-k2-01).<\/p>\n

Lausanne intestinal failure estimation (LIFE) score<\/h3>\n

The gastrointestinal failure score of patients was calculated on the seventh day after surgery in accordance with LIFE score estimation, as described previously65<\/a><\/sup>. Briefly, the evaluation criteria encompass various parameters, including levels of intra-abdominal pressure, lactate, gastric residue, enteral nutrition, motility, and bowel sounds.<\/p>\n

Human FABP2\/I-FABP immunoassay<\/h3>\n

Human blood samples were collected preoperatively and 12\u2009h after surgery using an anticoagulant (EDTA or heparin) and centrifuged at 400\u2009\u00d7\u2009g<\/i> for 15\u2009min to separate the upper layer of plasma. All samples were stored at \u221280\u2009\u00b0C before use. Human plasma FABP2\/I-FABP levels were determined using a human FABP2\/I-FABP Quantikine ELISA kit (DFBP20, R&D Systems) following the manufacturer\u2019s instructions, researchers being blinded to group allocations.<\/p>\n

D-lactate assay kit<\/h3>\n

Frozen human plasma samples were thawed at room temperature before detection. In accordance with the manufacturer\u2019s protocol, the colorimetric D-lactate assay kit (ab83429, Abcam) was used to measure D-lactate concentration in the plasma by researchers who were blinded to the group allocations.<\/p>\n

BMDM culture<\/h3>\n

To isolate BMDMs, the femur and tibiae of 7-8 week-old C57BL\/6\u2009J mice were flushed, the suspension centrifuged at 400\u2009\u00d7\u2009g<\/i> for 10\u2009min and the supernatant discarded. Subsequently, the precipitate was resuspended in DMEM (Gibco) supplemented with 10% FBS (Gibco) as well as 20% L929 conditioned mediam and cultured in a 37\u2009\u00b0C cell incubator. Seven days after differentiation, the cells were harvested for experiments. In the conditioned medium experiment, cells were cultured in organoid-derived conditioned medium or DMEM\/F12 as a control. In the MA stimulation experiment, cells were cultured with 4\u2009\u03bcM MA or an equal volume of double-distilled water (ddH2O) as a vehicle. Cells treated with siRNA for 24\u2009h before stimulation with MA were used as the negative control (NC) group. Cells were randomized into different groups as follows: (i) vehicle; (ii) MA; (iii) vehicle + siNC; (iv) vehicle+ siSOCS2; (v) MA + siNC and (vi) MA + siSOCS2.<\/p>\n

Untargeted and targeted metabolomics<\/h3>\n

Untargeted metabolomic analysis was performed by a Vanquish UHPLC system (Thermo Fisher Scientific) and an Orbitrap Q ExactiveTM<\/sup>HF-X mass spectrometer (Thermo Fisher Scientific). To obtain extracts, samples were homogenized and repeatedly centrifuged, and the final fecal extracts were aliquoted for metabolite profiling analyses. Raw data was generated using ultra-high performance liquid chromatography-mass spectrometry and processed by Compound Discoverer 3.1 (CD3.1, Thermo Fisher Scientific) for integration, normalization, and peak intensity alignment. Next, mzCloud, mzVault, and MassList databases were used to match the processed dataset. Principal component analysis and OPLS-DA were performed using normalized data, and VIP\u2009>\u20091 was considered as the threshold.<\/p>\n

Targeted quantitative metabolomics was conducted using TSQ Quantiva\u2122 (Thermo Fisher Scientific). The MA standard (L46691, Acmec, Biochemical, Shanghai, China), \u03b1KG standard (61234, Sigma-Aldrich) were dissolved in 4:1 methanol\/water (v\/v) to prepare the standard curve. The samples were homogenized with 80% methanol, sonicated for 3\u2009min, and centrifuged at 14,000\u2009\u00d7\u2009g<\/i> at 4\u2009\u00b0C for 10\u2009min. The supernatant obtained was filtered for subsequent experiments. A Prelude SPLC\u2122 sample preparation and liquid phase system (Thermo Fisher Scientific) was used to carry out Chromatographic separation, TSQ Quantiva (Thermo Fisher Scientific) was used to perform quantitative detection and the TraceFinder\u2122 software version 3.3 SP1 (Thermo Fisher Scientific) was used to complete data collection.<\/p>\n

RNA-seq<\/h3>\n

RNA was extracted as mentioned above. RNA sequencing was performed by Shanghai Biotechnology Corporation. Briefly, all RNA samples underwent quality control analysis using Fast QC (version 0.10.1). Sequencing libraries were prepared using a TruSeq Stranded Total RNA Library Prep Kit (15032612, Illumina, San Diego, CA, USA). CBot (HiSeq2500) was used to generate clusters and the HiSeq2500 sequencing platform (Illumina) was used to generate pair-end 150\u2009bp reads. Valid clean data were obtained using Trimadap to filter out unqualified sequences, while HISAT2 was used for reference genome alignment. Based on HISAT2 alignment results, transcripts were reconstructed using StringTie (John Hopkins University, Baltimore, MD, USA), and expression levels of all genes in each sample were calculated. The computation of differential gene expression was completed by the negative binomial model implemented in the Bioconductor package, edgeR. A fold change (FC)\u2009\u2265\u20092 or FC\u2009\u2264\u2009-2 with a q-value\u2009<\u20090.05 was considered significant.<\/p>\n

siRNA transfection<\/h3>\n

Mouse siRNA targeting SOCS2 and negative control siRNA were designed and constructed by Ribo Targets (Guangzhou, China). Transfection was performed via Lipofectamine 3000 (L3000015, Invitrogen), following the manufacturer\u2019s instructions. The efficiency of SOCS2 knockdown was assessed using qRT-PCR and western blotting.<\/p>\n

Macrophage isolation<\/h4>\n

Donor mice were gavaged with MA for 5 consecutive days and subjected to intestinal I\/R injury for 36\u2009h. Then the mice were sacrificed and the spleen was harvested. Spleen immune cell was isolated as described above and macrophages were further isolated by using magnetic bead separation methods. In short, determine the cell number in the single cell suspension and centrifuge cell suspension. Incubate the cell pellet with anti-F4\/80 microbeads (130-110-443, Miltenyi Biotec, Germany) according to the manufacturer\u2019s instructions.<\/p>\n

Adoptive transfer<\/h4>\n

Recipient mice underwent intestinal I\/R injury were injected intravenously with 2-3 \u00d7 10^6 macrophages immediately after the clamp was released.<\/p>\n

Statistical analysis<\/h3>\n

Statistical analyses were performed using GraphPad Prism (version 8.3.0) software. Two-tailed log-rank test was used for the Kaplan-Meier survival curve. The Shapiro-wilk test is used to determine whether the data is normally distributed. For normally distributed data, two-tailed student\u2019s t<\/i> test was used to compare the means of two independent groups. One-way ANOVA and Two-way ANOVA, followed by Tukey\u2019s multiple comparisons test, were used to compare the means of more than two groups. For non-normally distributed data, the nonparametric method such as Mann\u2013Whitney test was used. All data are expressed as mean\u2009\u00b1\u2009standard deviation. P\u2009<\u20090.05 was considered statistically significant. For further statistical details, refer to individual figure legends.<\/p>\n

Reporting summary<\/h3>\n

Further information on research design is available in the Nature Portfolio Reporting Summary<\/a> linked to this article.<\/p>\n