Inflammation triggers ILC3 patrolling of the intestinal barrier

An orchestrated cellular network, including adaptive lymphocytes and group 3 innate lymphoid cells (ILC3s), maintains intestinal barrier integrity and homeostasis. T cells can monitor environmental insults through constitutive circulation, scanning tissues and forming immunological contacts, a process named immunosurveillance. In contrast, the dynamics of intestinal ILC3s are unknown. Using intravital imaging, we observed that villus ILC3s were largely immotile at steady state but acquired migratory ‘patrolling’ attributes and enhanced cytokine expression in response to inflammation. We showed that T cells, the chemokine CCL25 and bacterial ligands regulated intestinal ILC3 behavior and that loss of patrolling behavior by interleukin-22 (IL-22)-producing ILC3s altered the intestinal barrier through increased epithelial cell death. Collectively, we identified notable differences between the behavior of ILC3s and T cells, with a prominent adaptation of intestinal ILC3s toward mucosal immunosurveillance after inflammation.

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Sample sizes were derived from initial preliminary experiments and from a principle of repeating all in vivo experiments at least twice to ensure experimental reproductibilty. No statistical methods were used to pre-determine samples sizes. For intravital imaging experiments, external parameters were controlled (temperature, oxygen), experimental bias was limited (no sample preparation besides surgery), multiple XY positions could be simultaneously acquired and the effect size was large -therefore a small sample size (n=3 in general) was powerful enough. For flow cytometry and Biomark experiments that involved sample preparation, sample size was of n=4 minimum (and in general > n=6) and this sample size was powerful enough too.
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Randomization Experimental groups were not randomized. Mice were grouped according to genotype and all experiments were performed with age-and sex-matched littermates. The sex-specific histocompatibility was observed for adoptive transfer.

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Investigators were not blinded during the acquisition of the data and analysis because 1) the investigator analyzing data was also collecting it 2) experimental groups could be determined from the data (e.g. images from intravital imaging where adoptive transfer could be deducted from CFP+ T cells that could be visualized on images).

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Mice were housed and bred in dedicated in animal facilities of the Institut Pasteur (12h light/dark cycle; 22+/-2°C) . All mice were used on a C57BL/6J background. Eight to fourteen weeks-old male and female animals, except for experiments on BM chimeric mice which nvolved sixteen weeks-old males at the time of analysis.

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was washed in PBS (Gibco) to eliminate mucus, cut into 1-2 cm pieces and intestinal epithelial cells were eliminated by shaking incubation in complete medium containing 5 mM EDTA (Invitrogen) for 20 min at 37°C. Subsequently, the intestinal tissue was minced and incubated twice in a digestion solution containing 0.5 mg Collagenase VII (Sigma-Aldrich) for 15 min at 37°C in a shaking incubator to isolate the lamina propria lymphocytes (LPL). LPL were filtered through a 40-μm cell strainer and kept in complete medium for downstream analysis.

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All the samples were acquired on a custom-configurated LSR Fortessa (BD Biosciences) or sorted using a custom-configurated FACSAria III (BD Biosciences).

Cell population abundance
The purities of sorted ILC3 were more than 98 %, as determined by flow cytometry analysis on the sample containing purified ILC3.

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