Pharmacological autophagy modulation bidirectionally regulates IL-17A and IL-22 production in intestinal ILC3 subsets of NEC mice

Experimental Design
Core Objective: Flow cytometry was performed to evaluate the effects of pharmacological autophagy modulation (autophagy inhibition by 3-Methyladenine, autophagy activation by Rapamycin) on the effector cytokine production of intestinal lamina propria total group 3 innate lymphoid cells (ILC3s) and double-negative (DN) ILC3 subsets in mice with necrotizing enterocolitis (NEC), by quantifying the percentage of IL-17A-producing and IL-22-producing cells within each ILC3 subset.
Animal Model:Wild-type mice on a C57BL/6 background were used in this study. All mice were subjected to standardized experimental NEC induction prior to pharmacological intervention and sample collection for flow cytometry detection.
Experimental Groups (3 groups total, fully matched to the figure):
DMSO group: Vehicle control group (NEC-induced mice, treated with DMSO vehicle)
3MA group: Autophagy inhibition group (NEC-induced mice, treated with 3-Methyladenine (3MA), a classical autophagy inhibitor)
Rapamycin group: Autophagy activation group (NEC-induced mice, treated with Rapamycin, a classical autophagy activator)
Detection Targets (fully matched to the figure axes):The percentage of cytokine-producing cells within ILC3 subsets in the intestinal lamina propria, including:
Percentage of IL-17A⁺ cells within total ILC3s
Percentage of IL-17A⁺ cells within DN ILC3s
Percentage of IL-22⁺ cells within total ILC3s
Percentage of IL-22⁺ cells within DN ILC3s
Biological Replicates: 7 biological replicates (individual mice) per group
Statistical Analysis: One-way ANOVA with Tukey’s post-hoc test was applied for multiple group comparisons. Statistical significance was defined as P < 0.05.
Flow cytometry data were acquired using a CytoFLEX S flow cytometer (Beckman Coulter). The following fluorochrome-conjugated antibodies were used for cell surface and intranuclear staining in this study:NKp46-FITC, Lin-APC-Cy7, live/dead-BV510, CD4-Pacific Blue (PB), CD90.2-PE-Cy7, IL17A/IL22-PE, CCR6-APC, and RORγt-PerCP-Cy5.5.