Activation of DR3 signaling causes loss of ILC3s and exacerbates intestinal inflammation

TNF-like ligand 1 A (TL1A) and death receptor 3 (DR3) are a ligand-receptor pair involved in the pathogenesis of inflammatory bowel disease. Group 3 innate lymphoid cells (ILC3s) regulate intestinal immunity and highly express DR3. Here, we report that activation of DR3 signaling by an agonistic anti-DR3 antibody increases GM-CSF production from ILC3s through the p38 MAPK pathway. GM-CSF causes accumulation of eosinophils, neutrophils and CD11b+CD11c+ myeloid cells, resulting in loss of ILC3s from the intestine in an IL-23-dependent manner and exacerbating colitis. Blockade of GM-CSF or IL-23 reverses anti-DR3 antibody-driven ILC3 loss, whereas overexpression of IL-23 induces loss of ILC3s in the absence of GM-CSF. Neutralization of TL1A by soluble DR3 ameliorates both DSS and anti-CD40 antibody-induced colitis. Moreover, ILC3s are required for the deleterious effect of anti-DR3 antibodies on innate colitis. These findings clarify the process and consequences of DR3 signaling-induced intestinal inflammation through regulation of ILC3s.

A full description of the statistics including central tendency (e.g. means) or other basic estimates (e.g. regression coefficient) AND variation (e.g. standard deviation) or associated estimates of uncertainty (e.g. confidence intervals) For null hypothesis testing, the test statistic (e.g. F, t, r) with confidence intervals, effect sizes, degrees of freedom and P value noted

Software and code
Policy information about availability of computer code Data collection Raw data were analyzed by FlowJo V7 and V10 for flow cytometric data, by ImageJ for immunofluorescence, by HiSeq 2000 System for RNA-seq data.

Data analysis
Statistical analyses were performed with Graphpad Prism 5. For RNA-seq data, reads were mapped to Mouse Genome Assembly GRCm38.p5 by STAR v2.5. Gene and isoform expression quantification was called by RSEM v1.2 with default parameters on GENCODE mouse M16 gene annotation file. Differential expression analysis was performed by Bioconductor package edgeR v3.18.1. Heatmap was generated with software HemI 1.0.
For manuscripts utilizing custom algorithms or software that are central to the research but not yet described in published literature, software must be made available to editors/reviewers upon request. We strongly encourage code deposition in a community repository (e.g. GitHub All studies must disclose on these points even when the disclosure is negative.

Sample size
For all experiments in this study, at least 3 samples per group were used. For most experiments, 5-12 mice were included in each group. For experiments using small sample sizes, littermate mice were used and the data were from at least two independent experiments.
Data exclusions No data were excluded in this study.

Replication
Data were pooled from at least two independent experiments. Consistent difference (or no difference) was observed, as indicated by the average value of each group, from at least two independent experiments. And statistical analyses were further performed based on the pooled data.
Randomization Littermate mice were randomly grouped into control and treatment groups for all experiments in this study.

Blinding
Data were collected by the same person carrying out the expriments. Two major authors worked individually and collaboratively on the project. Therefore, the data were not collected in a blinded manner. However, histological scoring has been performed double-blindedly.

Behavioural & social sciences study design
All studies must disclose on these points even when the disclosure is negative.

Study description
Briefly describe the study type including whether data are quantitative, qualitative, or mixed-methods (e.g. qualitative cross-sectional, quantitative experimental, mixed-methods case study).

Data collection
Provide details about the data collection procedure, including the instruments or devices used to record the data (e.g. pen and paper, computer, eye tracker, video or audio equipment) whether anyone was present besides the participant(s) and the researcher, and whether the researcher was blind to experimental condition and/or the study hypothesis during data collection.

Timing
Indicate the start and stop dates of data collection. If there is a gap between collection periods, state the dates for each sample cohort.

Data exclusions
If no data were excluded from the analyses, state so OR if data were excluded, provide the exact number of exclusions and the rationale behind them, indicating whether exclusion criteria were pre-established.

Location
State the location of the sampling or experiment, providing relevant parameters (e.g. latitude and longitude, elevation, water depth).

Access and import/export
Describe the efforts you have made to access habitats and to collect and import/export your samples in a responsible manner and in compliance with local, national and international laws, noting any permits that were obtained (give the name of the issuing authority, the date of issue, and any identifying information).

Describe any disturbance caused by the study and how it was minimized.
Reporting for specific materials, systems and methods

Authentication
Describe the authentication procedures for each cell line used OR declare that none of the cell lines used were authenticated.

Mycoplasma contamination
Confirm that all cell lines tested negative for mycoplasma contamination OR describe the results of the testing for mycoplasma contamination OR declare that the cell lines were not tested for mycoplasma contamination.

Commonly misidentified lines (See ICLAC register)
Name any commonly misidentified cell lines used in the study and provide a rationale for their use.

Palaeontology Specimen provenance
Provide provenance information for specimens and describe permits that were obtained for the work (including the name of the issuing authority, the date of issue, and any identifying information).

Specimen deposition
Indicate where the specimens have been deposited to permit free access by other researchers.

Dating methods
If new dates are provided, describe how they were obtained (e.g. collection, storage, sample pretreatment and measurement), where they were obtained (i.e. lab name), the calibration program and the protocol for quality assurance OR state that no new dates are provided.
Tick this box to confirm that the raw and calibrated dates are available in the paper or in Supplementary Information.

Animals and other organisms
Policy information about studies involving animals; ARRIVE guidelines recommended for reporting animal research

Laboratory animals
Wild-type or genetically engineered mice with the C57BL/6 background have been used in this study. Littermate mice were used for in vivo studies. Specifically, female litteramtes were used for DR3-Fc-treatment experiments. And both male and female mice have been used in experiments with a-DR3 injection.

Wild animals
The study didn't involve wild animals.

Field-collected samples
This study didn't involve samples collected from the feild.

April 2018
collected by the heart punctures. Mononuclear cells from the blood were isolated from interface of Ficoll-Paque density gradient centrifugation at 2500 rpm for 20 min at room temperature.
Gating strategy Tick this box to confirm that a figure exemplifying the gating strategy is provided in the Supplementary

Normalization
If data were normalized/standardized, describe the approach(es): specify linear or non-linear and define image types used for transformation OR indicate that data were not normalized and explain rationale for lack of normalization.

Normalization template
Describe the template used for normalization/transformation, specifying subject space or group standardized space (e.g. original Talairach, MNI305, ICBM152) OR indicate that the data were not normalized.

Noise and artifact removal
Describe your procedure(s) for artifact and structured noise removal, specifying motion parameters, tissue signals and physiological signals (heart rate, respiration).

Volume censoring
Define your software and/or method and criteria for volume censoring, and state the extent of such censoring.

Statistical modeling & inference
Model type and settings