Panel A shows two scatter bar plots comparing the frequency and mean fluorescence intensity of mitoSOX positive small intestine lamina propria ILC3s from Lingo4 fl fl and Lingo4 ILC3 conditional knockout mice, where the y-axis represents percentage and mean fluorescence intensity of mitoSOX positive ILC3s. Panel B shows two scatter bar plots of the frequency and mean fluorescence intensity of BODIPY 581 slash 591 C11 positive cells in small intestine lamina propria ILC3s, where the y-axis represents percentage and mean fluorescence intensity of lipid peroxidation. Panel C shows two scatter bar plots depicting the frequency and mean fluorescence intensity of phosphorylated STAT3 positive small intestine lamina propria ILC3s, where the y-axis represents percentage and mean fluorescence intensity of phosphorylated STAT3 positive ILC3s. Panel D shows representative flow cytometry plots and scatter bar plots of Annexin V and 7 aminoactinomycin D staining, where the y-axis represents the percentage of healthy, early apoptotic, late apoptotic, and necrotic ILC3s. Panel E shows a curve plot with data points of body weight variation over days post infection with Citrobacter rodentium, where the x-axis represents days post infection and the y-axis represents body weight variation in percent. Panel F shows scatter plots of fecal Citrobacter rodentium burden at day 10 and day 20 post infection, where the y-axis represents colony forming units per gram. Panel G shows flow cytometry plots and a scatter bar plot of the absolute number of colonic ILC3s, where the y-axis represents number of ILC3s. Panel H shows a scatter bar plot of the absolute numbers of ILC3 subsets, where the y-axis represents number of NKp46 positive, CCR6 positive, and double negative ILC3s. Panel I shows flow cytometry plots and scatter bar plots of cytokine producing colonic ILC3s at day 10 post infection, where the y-axis represents percentage of interleukin 22 positive, interleukin 17 positive, interferon gamma positive, tumor necrosis factor alpha positive, and granulocyte macrophage colony stimulating factor positive ILC3s.
Impaired ILC3 function is retained in Lingo4 ΔILC3 mice. (A) Frequency and MFI of mitoSOX+ siLP ILC3s from Lingo4fl/fl and Lingo4ΔILC3 mice. Mean ± SEM, n = 8–9; two experiments combined. (B) Frequency of BODIPY 581/591 C11–positive cells and MFI in Lingo4fl/fl and Lingo4ΔILC3 siLP ILC3s. Mean ± SEM, n = 8–9; two experiments combined. (C) Frequency and MFI of pSTAT3+ siLP ILC3. Mean ± SEM, n = 5. (D) Representative flow cytometry plots and frequency of Annexin-V and 7AAD staining of siLP ILC3. Mean ± SEM, n = 5. (E and F) Body weight variation (E) and fecal C. rodentium burden at 10 and 20 d.p.i. (F) of Lingo4fl/fl and Lingo4ΔILC3 mice during C. rodentium infection. Mean ± SEM, n = 6. (G–I) Representative plots and absolute numbers of colonic ILC3s (G), ILC3 subsets (H), and cytokine production by colonic ILC3 at 10 d.p.i. Mean ± SEM, n = 4. ILC3s were defined as live lymphocyte-sized Lin– (CD3− CD19−) Thy1.2hi CD45int cells. (A–I) Statistical significance was determined using unpaired two-tailed t test or one-way ANOVA with Tukey’s post hoc test. *P < 0.05; **P < 0.01; ***P < 0.001, ****P < 0.0001.