所有实验均在6-16周大的小鼠上进行。给定菌株的小鼠被随机分为不同的组，研究中都包括男性和女性。治疗和研究终止是由两个或多个实验者进行的，在结果评估过程中盲目进行了盲目，通过将动物标识符转换为样本处理和分析期间的数量。C57BL/6J（B6J，股票号000664）和DCAS9-KRAB（库存号030000）小鼠是从杰克逊实验室获得的；OT-1（库存号003831），OT-II（库存号004194），MyD88 - / - （参考文献32），TLR2 - / - （库存号004650），CD45.1，CD45.1（库存编号002014）和tlr4 - / - / - cop ref ref。ID2CREERT2; ROSA26EYFP小鼠34，IL5-转基因小鼠35和IFNGR2FL/FL MICE36先前已被描述。通过Riken发育生物学中心（登录号CDB0631K）和ST2 - / - （参考文献38）小鼠获得了IL33 - / - （参考文献37），并已被反向交叉到C57BL/6J背景上。在EPX启动子40（EO-CRE）下表达CRE的小鼠缺陷小鼠（Phil）和小鼠获自J. J. Lee。除非有指定，否则可随意提供食物和水。所有老鼠都处于B6J背景中，并以12小时的光线为12小时-12小时的黑暗时间表。在经认可的动物设施中，在没有特定的病因条件下饲养并饲养小鼠。饲养无菌小鼠并将其维持在柔性膜隔离器中的敞开式笼子中，并提供HEPA过滤的空气，以及高压灭菌的食物和水和水。在实验终点下，小鼠通过增加的二氧化碳浓度安乐死。苏黎世大学和伯尔尼大学的所有实验程序均按照瑞士联邦法规进行，并由州兽医办公室和/或根据欧洲社区理事会指令（86/609/EEC）批准，捷克国家准则，《捷克国家指南》，《 分子遗传学研究所的机构准则，并由动物护理委员会批准。 　　为期10天的治疗：7-8周龄的雌性和雄性小鼠（B6J）每周两次注射两次，用0.5 mg抗IL-5（BE0198 BIOXCELL，TREK5）或抗键孔的lim孔limolelole limpet limpet Heemocyanin型型控制Bioxcell，GR-20）或抗CCR3（BE0316克隆6S2-19-49）或抗脱氧化物过氧化物酶同种型对照（BE0088，Bioxcell，HRPN，HRPN）抗体在研究终点前10天。 　　连续10天用氨苄青霉素（1 g l-1; A0166 Sigma），万古霉素（500 mg l-1; A1839,0001; A1839,0001申请）连续10天治疗七个至八周龄的雌性小鼠（B6J）。如前所述41，在高压灭菌的饮用水中的Alfa aesar）。每周两次对水瓶进行监测和补充。 　　将6-12周龄的IL5-TG雌性和雄性小鼠的106个磁性脾嗜酸性粒细胞静脉注射到100 µL PBS中，将其注入CD45.1受体（8-12周龄大的雌性和雄性小鼠）中。注射后42小时收集器官。 　　用2.5％的DSS（w/v; 9011-18-1，MP生物医学）在高压灭菌的饮用水中用2.5％的DSS（Phil，B6J和IL33 - / - ）进行了六至2周大的雌性和雄性小鼠（Phil，B6J和IL33 - / - ）。每周两次对水瓶进行监测和补充。 　　在感染后四个星期后，用幽门螺杆菌菌株PMSS1（107个菌落形成单位，CFU）口服六到二个星期大的雌性和雄性小鼠（IL5-TG和B6J）口服口服。如前所述23，PMSS1菌株是一种十二指肠溃疡患者的临床分离株，在马琼脂板上生长，然后是液体培养的23。通过光学显微镜通常评估培养物，以污染，形态和运动。C.啮齿动物：6-12周大的雌性和雄性小鼠（IL5-TG和B6J）用nalidixic-抗酸性的c. rodentium C. rodentium菌株ICC169（ATCC 51549，108 CFU）口服，并在感染后分析13天。生物发光的啮齿动物菌株ICC180（ICC169衍生物，纳利迪酸 - 酸和抗Kanamycin耐药）是G. M. Frankel的礼物，以前被描述为42。两种菌株均在琼脂板上生长（1.5％； A0927 Applichem），然后在抗生素补充的Luria Broth（Nalidixic Acid，50μgml-1; N4382 Sigma和/或Kanamycin，50μgml-l-ML-1211 SIGMA）中; N4382 Sigma和/或Kanamycin，50μgml-1; N4382 SIGMA和/或KANAMYCIN; N4382 SIGMA; N4382 SIGMA; N4382 SIGMA; 420311 SIGMA） 　　每隔一天，每隔一天将七个至八个周的雌性小鼠（MyD88 - / - 和B6J）注射，三只总剂量为0.5 mg rec-il-33（210-33，peprotech）和/或IFNγ（315-05-05，peprotech）或PBS对照。 　　用啮齿动物梭状芽孢杆菌或未感染的七个星期大的雌性和雄性小鼠（IL5-TG和B6J）被口服。在分析前四天，向小鼠注射EDU（每只小鼠2.5 mg，900584 Sigma）。 　　四个月大的雌性小鼠（B6J）在50 µL PBS内接受1 µg HDM提取物以进行敏化（第0天），然后每天用10 µg HDM在50 µL PBS中进行5天（第7-11天）进行10 µg HDM挑战。致敏后14天收集肺。 　　用单剂量的他莫昔芬（T5648 Sigma）挑剔六到二个星期的雌性和雄性小鼠（ID2CREERT2； ROSA26EYFP）。将他莫昔芬溶解在少量的100％乙醇（在50°C下预热），然后将其重悬于玉米油中（在50°C下预热）至最终浓度每只小鼠5 mg。注射后2小时，2和4天收集器官。 　　收集胃，结肠和小肠，清洁粪便，并纵向切割。用PSB洗涤器官，切成碎片（1-2厘米），并从小肠中取出佩耶的斑块。用洗涤缓冲液（2％BSA，100 U ML -1青霉素 - 链霉素和5 mM EDTA，在HBSS中，25分钟，37°C）将碎片洗涤两次。然后将组织在冷PBS中冲洗，并在37°C的完整培养基（10％FBS和100 U ML-1青霉素 - 链霉素（P0781 Sigma）中，含RPMI-1640）在15 mM Hepes中（H0887 Sigma），0.05m sigma），0.05 mg mg mg ml roch i（1015）250 U ML -1型IV（C5138 Sigma）和VIII型胶原酶（C2139 Sigma）（用于结肠和小肠），或500 U ML -1 IV型胶原酶（C5138 Sigma）（用于胃）。细胞通过70μm细胞过滤器，离心8分钟，并分层到40/80％的Percoll（17089101 Cytiva）梯度（18分钟，2,100G，20°C，无制动）。收集相间并在PBS中洗涤。 　　将肺灌注PBS，收集并切成碎片，然后在补充了500 U ML -1型IV胶原酶（Sigma）和0.05 mg ML -1 DNase I（Roche）的完整培养基中，在37°C下持续50分钟。然后通过70μm细胞滤网通过肺部，并用注射器柱塞网格。为了减少巨噬细胞污染（SIGLEC-F+），将细胞在37°C的完整RPMI培养基中铺板1小时。 　　血液通过2％BSA 5 mM EDTA PBS的尸体心脏穿刺取样。对于IL5-TG小鼠，将悬浮液分层在1119（1.119 g ML-1的密度为1.119 g ML-1; 11191 Sigma-Aldrich）上，并在800g下离心20分钟，并在PBS中洗涤相间。将红细胞在冰冷的蒸馏水中裂解30 s。 　　使用完整的RPMI培养基和23口径针头冲洗股骨和胫骨。收集含量，通过40μm细胞过滤器过滤，并将红细胞在冰冷的蒸馏水中裂解30 s。 　　使用注射器柱塞收集脾脏和淋巴结通过40μm细胞过滤器，并在冰冷的蒸馏水中裂解30 s。 　　用5 mL PBS用21尺寸的针头灌注腹膜腔，并将膨胀区域按摩30 s，以分散溶液。收集腹膜液体，并将细胞在37°C的完整rpmi培养基中铺板1小时，以去除粘附的细胞。 　　将肺灌注PBS，并分离出围核脂肪仓库，从而去除任何可见的性腺组织。将组织切成小块，并在完整的DMEM培养基中消化，并在37°C下补充了0.2 mg ml -1 liberase（05401020001 roche）和0.05 mg ml -1 dnase I（roche）50分钟。通过100μm细胞过滤器过滤悬浮液，并以1,000g离心10分钟。收集颗粒并在PBS中洗涤。 　　收集子宫，纵向切割并在PBS中洗涤。在洗涤缓冲液（2％BSA，100 U ML -1青霉素 - 链霉素和5 mM EDTA）中摇动碎片，在HBSS中，25分钟，37°C）。然后将组织在冷PBS中冲洗，并在37°C的完整培养基中消化50分钟，其中含有0.05 mg ml -1 DNase I（Roche）和0.2 mg ml -1 liberase（Roche）。细胞通过70μm细胞滤网，离心并用PBS洗涤。 　　除非指定，否则所有离心步骤均以500g的速度在10°C下进行8分钟。 　　Eosinophils of 6–12-week-old female and male mice (Il5-tg) were positively enriched using a PE anti-mouse Siglec-F antibody (562068 BD Biosciences; E50-2440) and anti-PE microbeads (130-042-401 Miltenyi Biotech), according to the manufacturer’s instructions.根据制造商的说明，使用抗CD45微粒（130-052-301 Miltenyi Biotech）阳性地富集了7-8周龄雌性小鼠（B6J）的免疫细胞。 　　磁丰富的Siglec-F+嗜酸性粒细胞（血液，脾，胃，结肠，小肠，小肠，脂肪组织，肺和子宫，肺和IL5-TG），总BM细胞（IL5-TG）或CD4+细胞（COLON，B6J）使用B BID分析（BD）单个-Cell Single（BD）。每个样品从三只小鼠汇总细胞。上面描述了组织加工和富集程序。通过流式细胞仪评估每种制剂以确定嗜酸性粒细胞活力，并在细胞纺丝和染色后进行形态学检查。根据制造商的协议，将嗜酸性粒细胞标记为样品标签（633793 BD小鼠单细胞多路复用试剂盒）。简而言之，对于每种条件，将106个细胞重悬于染色缓冲液（1％BSA，PBS中的1％EDTA）中，并在室温下与各自的样品标签孵育20分钟。然后将细胞转移到5-mL聚苯乙烯管中，用2 mL染色缓冲液洗涤两次，并在400克离心5分钟。将样品重悬于1 mL染色缓冲液中以进行计数。接下来，将10,000或20,000个细胞从多达4个条形码样品合并，总共60,000个细胞，并以400克离心5分钟。将沉淀重悬于650 bd样品缓冲液中，并在（20 u µl-1; AM2694 Thermo Fisher Scientific）和NxGEN RNase抑制剂（40 U µL-1; 30281; 30281-2 Lucigen）中添加1：1,000 superase。BD Rhapsody弹药筒每个有60,000个电池。根据制造商的建议（BD Biosciences），用BD Rhapsody Express单细胞分析系统分离单细胞。使用BD Rhapsody全转录组分析扩增试剂盒（633801 BD Biosciences）制备cDNA文库，后BD Rhapsody System mRNA MRNA全转录组分析（WTA）和样本标签库制备协议（BD Biosciences）。使用Qubit DSDNA HS套件的量子荧光计对最终库进行定量 （Q32851 Thermo Fisher Scientific）。用贴纸4200系统（5067-5592安捷伦技术）上的Agilent高敏性D5000测定法测量了库的大小分布。在具有Novaseq 6000 SP Reagent Kit化学的Novaseq 6000上以配对末端模式（2×75个周期）进行测序。 　　在用BCL2FASTQ v.2.2.20.0.422（Illumina）和质量控制的BCL文件进行反复进行插图之后，在带有默认参数的七个Bridges Genomics平台上处理了配对的SCRNA-SECEQ FASTQ文件。下游分析是在r v.4.1.0中进行的，包装Seurat v.4.0.3（参考文献43）。合并了所有Seurat对象（每个多路复用样品一个）并进行相同的质量过滤。分析中排除了少于200或超过2500个基因的细胞。在日志归一化后，将计数数据缩放为线粒体读取，并根据2,000个最大可变特征执行主成分分析（PCA）。使用50个主组件在合并的数据集上进行聚类和UMAP可视化，对于共享最近的邻居聚类算法的分辨率为0.3。根据标记基因表达，手动对簇进行注释。下游分析中排除了上皮和间质污染物以及不属于嗜酸性粒子谱系的免疫细胞簇。也排除了高线粒体基因的簇。通过亚置表达嗜酸​​性粒细胞标记的簇分析嗜酸性粒细胞空间。如上所述，对子集数据集进行了标准化，缩放和PCA。使用20个主组件进行聚类和UMAP可视化，对于共享最近的邻居聚类算法的分辨率为0.3。对于肺，子宫和脂肪组织数据集，用Harmony44和上皮基因（具有PCT.2 < 0.05) derived from excessive-cell-free RNA were removed from the counts. 　　To extract cluster markers, FindAllMarkers was executed with logfc.threshold and min.pct cut-offs set to 0.25. Top-ranked genes (by log fold change; logFC) were extracted for illustration. For differential gene expression, FindMarkers was applied with logfc.threshold and min.pct set to 0. Genes were subsequently filtered on the basis of Bonferroni-adjusted P < 0.05. Scores were computed with the AddModuleScore function. Genes used for the scores and signatures were manually curated from Gene Ontology (GO) terms and literature, and are listed in Supplementary Table 3. Cell-cycle scoring was performed with the CellCycleScoring algorithm from Seurat, using cell-cycle-related genes45. For gene-set enrichment analysis (GSEA), differentially expressed genes were pre-ranked in decreasing order by the negative logarithm of their P value, multiplied for the sign of their average logFC (in R, ‘- log(p_val)*sign(avg_log2FC)’). GSEA was performed on this pre-ranked list using the R package FGSEA (https://github.com/ctlab/fgsea/) with default parameters and the GO Biological Process database, made accessible in R by the package msigdbr (https://github.com/cran/msigdbr). The results were filtered for significantly enriched gene sets (Bonferroni-adjusted P < 0.05). 　　Trajectory inference was performed with Monocle 2.3.6 (refs. 19,46) in R v.3.6.3. After creating a Monocle object using ‘negbinomial.size()’ distribution and lowerDetectionLimit = 0.5, the analysis was performed using Seurat’s top 2,000 variable features as ordering genes. Dimensionality reduction was performed using the DDTree method. To visualize the eosinophil differentiation, cluster annotations were projected on the inferred trajectories. Trajectory alignment of the BM–blood–colon trajectories was performed by applying dynamic time warping as described previously22,47. The steady-state and C. rodentium-challenge trajectories were set as the reference and query, respectively. Differentially expressed genes were identifying by using a full model of ‘y ~ pseudotime*treatment’ and a reduced model of ‘y ~ pseudotime’. 　　Loom files were generated with velocyto48 and dynamical velocities were computed with scvelo20. Fate probabilities were computed with CellRank21 and plotted as pie charts (partition-based graph abstraction, PAGA). 　　Pathway activity was calculated across eosinophil subsets with PROGENy v.1.13.2 (ref. 49) with default parameters. Gene-regulatory activity was interrogated by applying SCENIC 1.2.4 (ref. 28) with default parameters. In brief, after expression matrix filtering (minCountsPerGene = 3*.01*ncol(exprMat), minSamples = ncol(exprMat)*.01), and computing correlation, GENIE3 was applied to infer potential transcription factor targets. Co-expression networks were then calculated, regulons were created and their activity was scored in cells. Regulon activities were visualized as cluster averages using the R package ComplexHeatmap (ref. 50). 　　Challenge, DSS and B6J datasets were integrated using Seurat’s anchoring-based integration method using the steady-state object as reference dataset (reference.reduction = “pca”, dims = 1:50). 　　Ligand–receptor interaction analysis was performed using the Python package CellPhoneDB (v.2.0.0, Python v.3.8.5) following instructions from the GitHub repository (https://github.com/Teichlab/cellphonedb). In brief, the annotated Seurat object of isolated lamina propria immune cells from DSS-treated B6J mice was used to test the expression of known ligand–receptor interactions from the public repository of CellPhoneDB. Gene symbols were first converted from mouse to human using the biomart R package (v.2.46.3). Mean values representing the average ligand and receptor expression of annotated clusters were calculated on the basis of the percentage of cells expressing the gene, and the gene-expression mean. To determine the significance of observed means, P values were calculated using a null distribution of means calculated for randomly permuted annotated cluster labels. An interaction was considered significant if P ≤ 0.05. Significant ligand–receptor interaction pairs between eosinophils and CD8+ T cells or CD4+ T cells were extracted, gene symbols were converted from human to mouse and their mean values were plotted using the plot_cpdb function from the ktplots R package (v.1.1.14) (https://github.com/zktuong/ktplots). 　　Statistical analysis and visualization were performed using R version 3.6.3 or 4.1.0. Statistical significance tests were performed as described in each figure legend. Unless stated otherwise, all tests were significant with Bonferroni-adjusted P < 0.05. Plots were generated with the R package ggplot2 (ref. 51). 　　For surface staining, cells were stained in PBS at 4 °C for 30 min with the fixable viability dye eFluor 780 (1:1,000, 65-0865-14 eBioscience) and a combination of the following antibodies (1:200, all from BioLegend; unless stated otherwise): anti-mouse CD45 BV650 (30-F11, 103151), CD11b BV510 (M1/70, 101263), MHC-II AF700 (M5/114.15.2, 107622), Ly6G Percp-Cy5.5 (1A8, 127616), CD4 PerCP (RM4-5, 100538), TCRβ PE-Cy7 (H57-597, 109222), TCRβ PE-Cy7 (H57-597, 109228), CD80 BV605 (1:100, 16-10A1, 104729), PD-L1 PE-Cy7 (1:100, 10F.9G2, 124314), CD31 PE (390, 102408), CD45.2 BV785 (1:50, 104, 109839), CD9 PE (MZ3, 124805), CD54 BV711 (YN1/1.7.4, 116143), CD63 PE (1:100, NVG-2, 143904), CD95 PE-Cy7(SA367H8, 152607), Siglec-E PE (M1304A01, 677104), SCA-1 AF488 (D7, 108116), SCA-1 AF700 (D7, 108142), C-kit BV605 (ACK2, 135121), CD11c APC-Cy7 (N418, 117323), CLEC12a PE (5D3, 143404), CD49d FITC (R1-2, 103605), CD16/32 FITC (S17012B, 101305), CD3e Percp-Cy5.5 (145-2C11, 100328), CD8a APC (53-6.7, 100712), NK1.1 Percp-Cy5.5 (PK136, 108727), B220 Percp-Cy5.5 (RA3-6B2, 103236), Ter119 Percp (TER-119, 116227), Gr1 Percp (RB6-8C5, 108427), CD34 AF647 (RAM34, 560230), Siglec-F BV421 (E50-2440, 552681 BD Biosciences), Siglec-F PE (E50-2440, 552126 BD Biosciences), CD125 PE (T21, 558488 BD Biosciences), CD275 (HK5.3, 50598582 eBioscience) and T1/ST2 FITC (1:100, DJ8, 101001F MD Bioproductos GmbH). For T cell intracellular cytokine staining, cells were incubated for 3 h 15 min in complete IMDM medium containing 0.1 μM phorbol 12-myristate 13-acetate (P-8139 Sigma) and 1 μM ionomycin (I-0634 Sigma) with 1:1,000 Brefeldin A (00-4506-51 eBioscience) and GolgiStop solutions (51-2092KZ BD Biosciences) in a humidified incubator with 5% CO2 at 37 °C. After surface staining, cells were fixed and permeabilized with the Cytofix/Cytoperm Fixation/Permeabilization Solution kit (512090KZ BD Biosciences) according to the manufacturer’s instructions. Cells were then stained for 50 min with anti-mouse IL-17A APC (TC11-18H10.1, 506916), IFNγ BV421 (XMG1.2, 505830) and TNF FITC (MP6-XT22, 506 304) all from Biolegend at 1:100. Fc block (anti-CD16/CD32, 101302 Affymetrix) was included to minimize nonspecific antibody binding. Total leukocyte counts were determined by adding countBright Absolute Counting Beads (C36950 Life Technologies) to each sample before analysis. Samples were acquired in a LSRII Fortessa or FACS AriaIII 5L (BD Biosciences). For high-dimensional spectral flow cytometry analysis, cells were acquired on Cytek Aurora 5L (Cytek Biosciences) following 50 min staining at 4 °C with the antibodies described in Supplementary Table 5. For the Click-iT Plus EdU Alexa Fluor 647 Flow Cytometry Assay Kit (C10419 Thermo Fisher Scientific), the staining protocol was followed according to the manufacturer’s instructions. BD FACSDiva Software (BD Biosciences) was used for data acquisition and cell sorting. 　　Flow cytometry data analysis was performed with FlowJo software (v.10.7.1 Becton Dickinson). Cell counts, relative cell frequencies or MFI were used to generate graphical plots in GraphPad Prism (v.9.1.1, GraphPad). High-dimensional flow cytometry data were compensated and exported with FlowJo software (v.10) and the resulting FCS files were uploaded into Rstudio (v.4.0.3 R software environment). UMAPs were generated on stochastically selected cells from each sample and FlowSOM metaclusterings were performed for all the exported events as described previously52. 　　All statistical analyses were performed with GraphPad Prism (v.9.1.1, GraphPad). Two-tailed unpaired Student’s t-tests were used for comparing two groups, and comparisons of more than two datasets were done using a one-way analysis of variance (ANOVA) with Tukey’s post-test. Differences were considered statistically significant when P < 0.05. 　　To generate mouse BM-derived eosinophils (BM-Eos), BM cell suspensions were seeded at a density of 106 cells per ml in RPMI-1640 medium supplemented with 20% heat-inactivated FBS, 25 mM HEPES (H0887 Sigma), 100 U ml−1 penicillin–streptomycin (P0781 Sigma), 2 mM glutamine (25030-024 Gibco), 1× NEAA (11140-035 Gibco), and 1 mM sodium pyruvate (11360070 Gibco). Cells were cultured in a humidified incubator with 5% CO2, 37 °C, and were supplemented with 100 ng ml−1 mouse SCF (250-03 PeproTech) and 100 ng ml−1 mouse FLT3-Ligand (250-31L PeproTech) from day 0 to day 4, followed by differentiation with 10 ng ml−1 mouse rec-IL-5 (215-15 PeproTech) until day 13, as described53. Half of the medium was replaced and the cell concentration was adjusted to 106 cells per ml every other day. On day 8, cells were collected and moved to new flasks to remove adherent contaminating cells. On day 13, the nonadherent cells were collected and washed with PBS. Eosinophils were sorted and purity was assessed by flow cytometry (higher than 95%). 　　Supernatant of cultured colonic explants (colon CM) was prepared by culturing mid-colon sections (around 0.3 cm) from 6–12-week-old female and male mice (B6J) in 300 µl complete RPMI medium in a humidified incubator with 5% CO2, 24 h at 37 °C. Flow-cytometry-purified eosinophils were magnetically isolated from blood and spleen (Il5-tg) or differentiated from the BM (B6J) and were kept in complete RPMI medium with recombinant mouse IL-5 (10 ng ml−1, PeproTech). Cells were seeded in round-bottom 96-well plates at a density of 2 × 105 cells per well (100 µl) and conditioned for 12 h at 37 °C with cell-free colon CM (1:10 or at the indicated doses) or the following cytokines: IL-22 (10 ng ml−1, 210-22 PeproTech), IL-25 (10 ng ml−1, 210-17E PeproTech), TNF (10 ng ml−1, 315-01A PeproTech) and IL-33 (20 ng ml−1 or at the indicated doses, PeproTech). The NF-κB inhibitor BAY11-7082 (B5556, Sigma) was added at a concentration of 5 μM and anti-IL-33 neutralizing antibody (AF3626, Biotechne) at 30 ng ml−1. To study granule mobilization, magnetically enriched splenic eosinophils (Il5-tg) were treated overnight with colon CM (1:10) and flow-cytometry-sorted A-Eos were conditioned with IFNγ (20 ng ml−1, PeproTech) for 90 min. 　　Flow-cytometry-purified BM-Eos (B6J) or magnetically enriched colonic, splenic and blood eosinophils (Il5-tg) from 6–12-week-old female and male mice were used for the assay. BM-Eos were conditioned overnight with colon CM (1:10) at 37 °C. Eosinophils were washed with PBS and transferred to a white flat-bottom 96-well plate (Corning) in antibiotic-free RPMI-1640 medium supplemented with 10% FBS and mouse IL-5 (10 ng ml−1, PeproTech). A total of 108 bioluminescent C. rodentium bacteria (at exponential phase, optical density at 600 nm (OD600 nm) = 1–1.5) was added to each well and luminescence was measured after 60 min on an Infinite 200 PRO plate reader (TECAN). 　　Flow-cytometry-purified BM-Eos (B6J) or magnetically enriched splenic eosinophils (Il5-tg) or A-Eos and B-Eos sorted from the GI tract (Il5-tg) were isolated from 6–12-week-old female and male mice. BM-Eos or spleen-derived eosinophils were conditioned overnight with colon CM (1:10) or treated with recombinant mouse IFNγ (10 ng ml−1, PeproTech) and/or IL-33 (20 ng ml−1, PeproTech), as indicated. Naive CD4+ T cells were isolated from the lymph nodes of 6–12-week-old female and male mice (B6J), enriched with the MojoSort Mouse CD4 Naïve T Cell Isolation Kit (480040 BioLegend) and purified by flow cytometry. T cells were labelled with the CellTrace CFSE Cell Proliferation Kit (C34554 Thermo Fisher Scientific) following the manufacturer’s instructions. T cells were then activated by CD3/CD28 T-activator Dynabeads (11131D Gibco) and co-cultured with eosinophils at a 1:1 ratio (2 × 105 total) for 4 days at 37 °C in complete RPMI medium supplemented with 10 ng ml−1 recombinant mouse IL-5 (PeproTech) and 20 ng ml−1 IL-2 (402-ML R&D). CFSE dilution was assessed by flow cytometry. 　　BM-Eos were isolated from 6–8-week-old female and male mice (B6J) and purified by flow cytometry. Eosinophils were conditioned overnight with colon CM, where indicated. Cells were washed in PBS and loaded with 300 n ml−1 of ovalbumin (OVA) residues 257–264 (S7951 Sigma) or 323–339 (O1641 Sigma) for 6 h in complete RPMI medium supplemented with 10 ng ml−1 recombinant IL-5 (PeproTech). T cells were sorted by flow cytometry and labelled with CellTrace CFSE Cell Proliferation Kit (C34554 Thermo Fisher Scientific) following the manufacturer’s instructions. OT-I CD8+ and OT-II CD4+ T cells were obtained from the lymph nodes of 8–12-week-old female and male mice (OT-I and OT-II, respectively). T cells were co-cultured with eosinophils at a 1:1 ratio (2 × 105 total) for 4 days at 37 °C in complete RPMI medium supplemented with 10 ng ml−1 recombinant mouse IL-5 (PeproTech) and 20 ng ml−1 IL-2 (402-ML R&D). CFSE dilution was assessed by flow cytometry. 　　The RNA from cultured BM-Eos (B6J) or A-Eos and B-Eos sorted from the small intestine (Il5-tg) was isolated using the Direct-zol RNA MicroPrep kit (R2062 Zymo Research), whereas the RNA from magnetically enriched colonic, splenic and blood eosinophils from 6–12-week-old female and male mice (Il5-tg) was isolated using the RNeasy Mini kit (74106 QIAGEN). Both isolations were performed according to the manufacturer’s instructions, including the on-column DNase 1 digestion step. Complementary DNA synthesis was performed using Superscript III reverse transcription (18080-044 QIAGEN). Gene expression was measured on a CFX384 Touch Real-Time PCR system (Bio-Rad, Second Derivative Maximum method analysis with high-confidence algorithm) by TaqMan Gene Expression Assays (4331182 Applied Biosystems by Thermo Fisher Scientific): Cxcl2 (Mm00436450_m1), Hprt (Mm03024075_m1), Gapdh (Mm99999915_g1), Cd274 (Mm03048248_m1), Cd80 (Mm00711660_m1), Ahr (Mm00478932_m1), Nfkb1 (Mm00476361_m1), Nfkb2 (Mm00479807_m1), Rela (Mm00501346_m1), Tnfa (Mm00443258_m1), Il1b (Mm00434228_m1) and Ptgs2 (Mm00478374_m1). Gene-expression levels for each sample were normalized to Hprt or Gapdh expression. Mean relative gene expression was determined, and the differences calculated using the 2ΔC(t) method. 　　BM-Eos were isolated from seven-to-eight-week-old female and male mice (B6J), differentiated and purified by flow cytometry. Cells were plated at a density of 5 × 105 cells per well (250 µl) and conditioned overnight with recombinant IL-33 (20 ng ml−1 PeproTech) and/or IFNγ (15 ng ml−1 PeproTech). RNA isolation was performed with the RNeasy Mini kit (74106 QIAGEN) according to the manufacturer’s instructions, including the on-column DNase 1 digestion step. RNA quality was assessed by Tapestation (Agilent). Library preparation was performed with the Illumina TruSeq RNA Kit. RNA sequencing was performed on the Illumina Novaseq 6000 (200 Mio reads), single-end read 100 bp. Reads were quality-checked with FastQC. Read alignment to the reference genome Mus_musculus.GRCm39 and read count was performed on the Support Users for SHell script Integration (SUSHI) framework54, with the RSEMApp application. Filtering and differential expression testing were performed with edgeR (ref. 55). The package pheatmap (ref. 56) was used to generate heat maps. 　　The colon of 7–8-week-old female and male mice (B6J) was dissected out, flushed in PBS and fixed 3 h in PFA (4% in PBS) at 4 °C, followed by overnight incubation in sucrose (30% w/v in 4% PFA) at 4 °C. Tissue was embedded in Tissue-Tek OCT Compound (Sakura, 4583) and stored at −80 °C. Tissue from three or four mice was cryosectioned (8 µm) onto the same microscope slide, washed in PBS and incubated for 1 h in blocking solution (2.5% BSA, 5% heat-inactivated normal goat serum, 0.1% Tween-20 in PBS) at room temperature. Slides were incubated overnight in blocking solution with the following primary antibodies (1:100): rat anti-mouse Siglec-F (E50-2440, 552126 BD Biosciences), Armenian hamster anti-mouse CD80 (16-10A1, 104729 Biolegend) and rabbit anti-mouse pNF-κB p65 (Ser536) (93H1,3033S Cell Signalling). After washing three times with PBST (0.1% Tween in PBS), the following secondary antibodies were added (1:400 in blocking solution) to the slides for 1 h at room temperature: AlexaFluor goat anti-rat 594 (A-11007), AlexaFluor goat anti-hamster 647 (A-21451) and AlexaFluor goat anti-rabbit 488 (A-11008), all from Thermo Fisher Scientific. Slides were washed four times for 5 min with PBST, and DAPI (D9542 Sigma, 1:1,000) was added to the third washing step. Slides were mounted in Prolog Gold (P36930 Invitrogen) and imaged on a Nikon Ti2-E inverted microscope, equipped with CrestOptics X-Light v3 confocal disk unit, Lumencor Celesta lasers and Photometrics Kinetix camera. 　　The microarrays CO245 and CO246 were obtained from TissueArray.Com. Deparaffinized sections were subjected to antigen retrieval in 2.4 mM sodium citrate and 1.6 mM citric acid, pH 6, for 25 min in a steamer. Sections were washed with PBST and blocked for 1 h at room temperature in blocking buffer (5% BSA, 5% heat-inactivated normal goat serum in PBST). Slides were incubated overnight at 4 °C with the following primary antibodies (1:100, in blocking buffer): mouse anti-human MBP (BMK-13, anti-human MBP (BMK-13, MCA5751 Bio-Rad) and rabbit anti-human PD-L1 (E1L3N, 13684S Cell Signalling). After washing three times with PBST (0.1% Tween in PBS), the following secondary antibodies were added (1:400 in blocking solution) to the slides for 1 h at room temperature: AlexaFluor goat anti-rabbit 594 and AlexaFluor goat anti-mouse 647 (Thermo Fisher Scientific). DAPI staining, mounting and imaging were performed as above. 　　A total of 105 FACS-enriched spleen, blood and GI-tract-derived eosinophils (Il5-tg) from 7–8-week-old female and male mice were resuspended in 100 µl 5% FCS-supplemented RPMI medium and cytospun for 5 min at 50g into a funnel. Slides were air-dried for 30 min, fixed with ice-cold methanol for 5 min and then left to air dry overnight. Slides were washed, incubated for 1 h in blocking solution and stained overnight at 4 °C with mouse anti-EPX antibody (MM25-82.2.1 1:200, provided by E. A. Jacobsen), followed by 1-h incubation at room temperature with AlexaFluor goat anti-mouse 647. DAPI staining, mounting and imaging were performed as above. EPX staining intensity was quantified across the cell diameter in Fiji (MultiPlot) for 15 cells per condition. 　　The cores used for quantification as well as patient data are available in Supplementary Table 2. Cores were chosen on the basis of the presence of colonic epithelium. ND files were imported in Imaris 9.6.0 and spot objects were created in the green (MBP) and red (PD-L1) channels separately (estimated XY diameter = 7 µm, estimated Z diameter = 4 um, quality filter > 6）。为了量化PD-L1和MBP的共表达，计算了绿色通道中每个斑点与红色通道中最近位置的距离。绿点（嗜酸性粒细胞），距离红斑距离 < 4 µm were considered as active eosinophils (co-expressing PD-L1). Green spots with distance to red spots > 4 µM被认为是基础嗜酸性粒细胞。然后，通过将活动的数量除以每个核心中的基底嗜酸性粒细胞数来计算活性与基础比。为了进行定位分析，在手动绘制的ROI中计算了人类和小鼠组织结肠隐窝中的活性与基础比，其中包括下部（基底）或上部（腔）三分之二。 　　将横向中部切片（0.5 cm）固定在缓冲10％的福尔马林溶液中，然后用石蜡嵌入。将部分用H＆E染色。考虑四类（每种评分为0-3）：上皮增生或损伤以及杯状细胞耗竭；白细胞浸润在固有层中；粘膜下炎症和水肿；受影响的组织区域。提出的最终分数（0-12）代表所有类别的总和。 　　如上所述，分离出了10-16周大的雌性和雄性小鼠（n = 27，dcas9-krab）的总共13亿BM干细胞（BMSC）。然后将BMSC分为两种重复，每个慢病毒都用独立扩增的全基因组CRISPR抑制库57（Addgene 83987）进行转导。转导五天后，富含BFP+ BMSC，并用重组IL-5（10 ng ml-1，peprotech）补充其培养基。在IL-5介导的分化六天后，将BM-EOS用结肠CM调节过夜（1:10）。对PD-L1+ CD80+嗜酸性粒细胞进行排序，提取基因组DNA，并将SGRNA靶向放大。用贴纸4200系统（5067–5592安捷伦技术）上的Agilent高敏性D5000测定法测量了库的大小分布。在Illumina NextSeq上以单端模式（75个周期）进行测序。读取读物用custadapt（参考文献58），并与bowtie2（参考文献59）对齐。Mageck（参考文献60）用于指导计数和配对测试。 　　从8-10周龄的雌性和雄性小鼠（B6J）中分离出BM-EOS，通过流式细胞仪进行区分和纯化。用结肠CM（1:10）或Rec-IL-33（20 ng ml-1 peprotech）调节细胞45分钟，然后在RIPA缓冲液（R0278 Sigma）中裂解，补充了2 mM钠钠钠钠酸钠（J60191.AE Herto Fisherscientific），热渔民，15 mmmmmmmmmmsod2.科学），10毫米氟化钠（447351000 Thermo Fisher Scientific）和1倍完整的蛋白酶抑制剂鸡尾酒（11836153001 Roche）。通过BCA测定法（23227 Pierce）测定蛋白质浓度，并使用10％丙烯酰胺凝胶通过SDS -PAGE分离蛋白质浓度，然后将其转移到硝基纤维素膜上（88018 Thermo Fisher Scientific）。用抗Vinculin（42H89L44，700062 Thermo Fisher Scientific），Phosho-P38 MAPK（Thr180/Tyr182，MA5-15218 Thermo Fisher Scientific）和磷酸p65（Ser536，93H1，3033 Cells Signal-signal Indroge）探测膜。 　　从在450μLRIPA裂解缓冲液（Thermo Fisher Scientific）中均质的结肠样品中提取蛋白质，并补充了Na3VO4（100 mM），NAF（10 mM）和蛋白酶抑制剂鸡尾酒（完整的，Mini蛋白酶抑制剂片剂，11836153001 ROCHE）。收集上清液并在4°C下以最大速度离心10分钟。用Pierce BCA蛋白测定试剂盒（23225，Thermo Fisher Scientific）对蛋白质浓度进行定量。在BD微型管中的血液中分离血浆（365968，BD）。根据制造商的说明，使用板覆盖​​过夜，并使用小鼠IL-33 ELISA试剂盒（88-7333-88 Thermo Fisher Scientific）来量化IL-33的结肠和血浆水平。 　　如上所述提取蛋白质。根据制造商的说明，使用LegendPlex MU TH17面板（7-PLEX）对IFNγ，IL-22和TNF的结肠和血浆水平进行定量。 　　将来自三个溃疡性结肠炎患者的新鲜冷冻结肠样品截至盖玻片上，并通过解决生物科学进行处理。 　　使用预审计的核模型和Flow_threshold 0.5，CellProb_threshold -0.2，使用细胞蛋白（v。2.0.4）（参考文献61）在DAPI图像中分割核。然后，使用Scikit-图像中的“ Expand_labels”功能通过10像素（1.38 µm）扩展核节段，然后将转录本分配给扩展的段。从分析中除去了少于三个分子或三个基因的片段。 　　为了避免分割的问题，我们使用了一种以转录本为中心的方法，在该方法中，我们使用特定标记基因的空间簇表示细胞类型并研究共定位。为此，使用2D坐标的Euclidean距离计算了CD4，Siglec8，CD8A，CD19，FOX3和FCN1的单个成绩单之间的距离。然后将层次聚类应用于距离矩阵，并具有平均连锁，以防止链条，并在5 µm的高度切割树（Stats r包中的Hclust）。然后，我们使用基于K-d-Tree的最近邻居搜索来识别彼此周围区域中群集中的簇，并在R函数“ NN2”（RANN v.2.6.1，SearchType ='radius'）中实现的预定的半径为10 µm，并具有足够的大k（k = 41）。该方法在O（m logM）时间内运行，并避免计算成千上万个对象的距离矩阵。最后，从所得的邻接矩阵中构造了一个邻域图，其中顶点（成绩单簇）如果不超过10 µm，则通过边缘连接。从该图中，计算了不同单元格类型之间的边缘数，并将其与从随机置换顶点标签（M = 1,000）的经验无效分布进行了比较。该方法考虑了组织组成和空间结构，并允许p值计算为p =（b+1）/（m+1），其中b是置换量在两种单元类型之间产生的边缘比观察到的置换总数更高的边缘的次数。这是针对每个载玻片和可能的细胞 - 细胞相互作用进行的，以得出一个分数，该分数代表特定相互作用显着的图像的比例，其中符号代表相互作用或回避的符号；参考文献采用了可视化。63。 　　实验工作流的示意图是使用Biorender.com的许可版本创建的。 　　有关研究设计的更多信息可在与本文有关的自然投资组合报告摘要中获得。