Panel A shows a schematic of the cohousing experimental setup along with a scatter bar plot of absolute numbers of different innate lymphoid cell subsets (ILC1, ILC2, ILC3) in the small intestine lamina propria of cohoused Lingo4 wild type, heterozygous, and knockout littermates, where the x-axis represents the cell types and the y-axis represents absolute cell numbers. Panel B shows representative flow cytometry plots and scatter bar plots of the frequencies of interleukin 22 positive and interleukin 17 positive ILC3s after mock or interleukin 23 stimulation, where the x-axis represents conditions and the y-axis represents percentage of cytokine positive cells. Panel C shows a treatment scheme with flow cytometry plots and scatter bar plots of ILC3 frequencies in non cohoused, antibiotic treated wild type and Lingo4 knockout mice, where the x-axis represents conditions and the y-axis represents percentage of ILC3s within CD45 positive cells. Panel D shows flow cytometry plots and scatter bar plots of interleukin 22 positive and interleukin 17 positive ILC3s after interleukin 23 stimulation in antibiotic treated mice, where the x-axis represents conditions and the y-axis represents percentage of cytokine positive cells. Panel E shows representative flow cytometry plots of ILC3s in wild type and Lingo4 knockout mice at different ages. Panel F shows a scatter bar plot of the number of ILC3s at different ages, where the x-axis represents age in weeks and the y-axis represents cell number. Panel G shows a scatter bar plot of the frequencies of interleukin 22 positive ILC3s following interleukin 23 stimulation at different ages, where the x-axis represents age in weeks and the y-axis represents percentage of cytokine positive cells. Panel H shows a principal coordinates analysis plot of Bray Curtis distances indicating beta diversity of fecal microbiota composition, where the x-axis represents principal coordinate 1 and the y-axis represents principal coordinate 2, and dot size reflects alpha diversity. Panel I shows stacked bar plots of the relative abundance of different bacterial families in the fecal microbiota, where the x-axis represents different mouse groups and the y-axis represents relative abundance at the family level.
Reduced ILC3 responses in Lingo4 −/− mice depend on gut microbiota. (A) Numbers of ILCs in siLP of cohoused Lingo4+/+, Lingo4+/−, and Lingo4−/− littermates. Mean ± SEM, n = 9–11; three experiments combined. (B) Representative flow cytometry plots and frequencies of IL-22+ and IL-17+ ILC3s after IL-23 stimulation. Mean ± SEM, n = 4; representative of three experiments. (C and D) Antibiotic depletion of microbiota. VNAM treatment scheme and frequencies of ILC3s in non-cohoused VNAM-treated WT and Lingo4−/− mice (C). IL-22+ and IL-17+ ILC3s after IL-23 stimulation (D). ILC3s were defined as live lymphocyte-sized Lin– (CD3− CD19−) Thy1.2hi CD45int cells. Mean ± SEM, n = 4; representative of two experiments. (E–G) Representative plots (E), numbers of ILC3s (F), and IL-22+ ILC3 frequencies following IL-23 stimulation (G) in WT and Lingo4−/− mice at different ages. ILC3s were defined as live lymphocyte-sized Lin− (CD3− CD19−) Thy1.2hi CD45int cells. Mean ± SEM, n = 4–5. (H and I) Fecal microbiota composition analysis of cohoused and non-cohoused WT and Lingo4−/− mice. Principal coordinates analysis (PCoA) of Bray–Curtis distances showing β diversity; dot size indicates α diversity (Shannon index) (H). Relative abundance at the family level (I). n = 4. (A–G) Statistical significance was assessed using unpaired two-tailed t tests or one-way ANOVA with Tukey’s post hoc test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.