Proportion of ILC3 subsets in NEC mice with autophagy deficiency and metabolic interventions

Experimental Design
Core Objective: Flow cytometry was performed to evaluate the effects of Atg5-mediated autophagy deficiency and metabolic interventions (lactate supplementation, etomoxir treatment) on the proportion and absolute number of total group 3 innate lymphoid cells (ILC3s) and double-negative (DN) ILC3 subsets in the intestinal lamina propria of mice with necrotizing enterocolitis (NEC).
Animal Model:Genetically modified mice on a C57BL/6 background were used in this study:
Atg5fl/fl mice: Wild-type control mice with intact Atg5-mediated autophagy function in RORγt+ cells (ILC3 lineage)
RorccreAtg5fl/fl mice: RORγt+ cell-specific conditional Atg5 knockout mice, with autophagy deficiency specifically restricted to the ILC3 lineage
All mice were subjected to standardized experimental NEC induction prior to intervention and sample collection.
Experimental Groups (4 groups total, fully matched to the figure):
Atg5fl/fl + DMSO: Vehicle control group (autophagy-intact ILC3s, vehicle treatment)
RorccreAtg5fl/fl + DMSO: Autophagy-deficient vehicle group (ILC3-specific autophagy deficiency, vehicle treatment)
RorccreAtg5fl/fl + Lactate: Metabolic intervention group (ILC3-specific autophagy deficiency, lactate supplementation)
RorccreAtg5fl/fl + Etomoxir: Metabolic intervention group (ILC3-specific autophagy deficiency, fatty acid oxidation inhibitor etomoxir treatment)
Detection Targets (fully matched to the figure axes):The proportion and absolute count of ILC3 subsets in the intestinal lamina propria, including:
Percentage of total ILC3s among total ILCs
Percentage of DN ILC3s among total ILCs
Absolute number of total ILC3s
Absolute number of 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: Lineage-FITC, CD45-APC-Cy7, CD4-BV510, CD127-Pacific Blue (PB), CD90.2-PE-Cy7, NKp46-PE, CCR6-APC, and RORγt-PerCP-Cy5.5.