Journal: Nature Communications
Article Title: TCF-1 and TOX regulate the memory formation of intestinal group 2 innate lymphoid cells in asthma
doi: 10.1038/s41467-024-52252-2
Figure Lengend Snippet: A In asthma remission, KLRG1 – , KLRG1 + IL-17RB – , KLRG1 + IL-17RB + subsets (marked as AR-KLRG1 – , AR-KLRG1 + IL-17RB – and AR-KLRG1 + IL-17RB + ) in total CD45 + lineage – CD90.2 + ST2 – ILC2s in siLP were sorted. KLRG1 + IL-17RB + ILC2 (marked as naive-KLRG1 + IL-17RB + ) were isolated from siLP of naive WT mice (5 ~ 7 × 10 5 for every subset, purity > 98%). Total RNA was isolated, and qRT-PCR was performed. Heat map of mRNA expression values of selected genes (see Fig. ). n = 6 for AR-KLRG1 – ILC2, AR-KLRG1 + IL-17RB – ILC2 and AR-KLRG1 + IL-17RB + ILC2, n = 3 for naive-KLRG1 + IL-17RB + ILC2. B – F Sorted CD45 + lineage – CD90.2 + NK1.1 – NKp46 – ST2 – KLRG1 + IL-17RB + ml-ILC2s were transfected with siRNAs targeting Tcf7 or Tox (si Tcf7 or si Tox ) or negative control (Negative Ctrl). Together with AR-KLRG1 – , AR-KLRG1 + IL-17RB – and naive-KLRG1 + IL-17RB + ILC2s (5 ± 10 3 per well), a combination of 200 ng/mL IL-25 and 200 ng/mL IL-33 were added every two days with IL-2 and IL-7 for 8 days. Knockdown efficiency of Tcf7 or Tox was confirmed (see Fig. ). B Cell expansion was calculated relative to day 0. C Protein levels of IL-13 in culture medium were detected by ELISA. n = 8 for ml-ILC2 Ctrl, ml-ILC2 Negative Ctrl, ml-ILC2 si Tcf7 and ml-ILC2 si Tox , n = 5 for AR-KLRG1 – ILC2, AR-KLRG1 + IL-17RB – ILC2 and naive-KLRG1 + IL-17RB + ILC2. D Heat map of mRNA expression values of selected genes (see Fig. ). n = 3. Expression of E CCR9 and F S1PR1 of all cells in each group were detected with flow cytometry. n = 3. G Flow chart of asthma mice model, BrdU administration, RNA interference and sample collection. On days 7–11, BrdU (0.8 mg/mice) was given intranasally daily. Rag1 – / – mice ( n = 5) were injected with Ambion in vivo si-negative control or indicated siRNAs on days 12–18, and were intranasally treated with 25 μg HDM on days 72-74. The percentage of BrdU + in ST2 – KLRG1 + IL-17RB + ILC2s in H siLP on day 71 and in ( I ) lungs on day 75 were detected with flow cytometry. J Flow chart of asthma mice model, adoptive transfer, RNA interference, and sample collection. Rag1 – / – mice were exposed with HDM and rested till AHR vanished as described in Fig. . Mice were injected with Ambion in vivo si-negative control, si Tcf7 or si Tox on days 16-18, and were intranasally treated with 25 μg HDM on days 19–21. For the groups of in vivo si Tcf7 +ml-ILC2 AT + HDM and in vivo si Tox +ml-ILC2 AT + HDM, mice were injected (i.v.) with 5 × 10 5 ml-ILC2s precultured with transfection of si-negative control on day 19 before HDM treatment; For the groups of in vivo si Tcf7 +si Tcf7 -ml-ILC2 AT + HDM and in vivo si Tox +si Tox -ml-ILC2 AT + HDM, mice were injected (i.v.) with 5 × 10 5 ml-ILC2s precultured with transfection of siRNAs on day 19 before HDM treatment. Twenty-four hours after the last HDM treatment, K airway resistance ( n = 3) and L IL-13 level in lung homogenates were measured ( n = 6). M Pulmonary histological changes were analyzed ( n = 6). Magnification: ×200, scale bar = 100 μm. Data was shown as Means ± SD. Results are representative of three independent experiments. Statistical comparison was conducted using unpaired one-way ANOVA with Dunnett’s test, except in ( B ) and ( K ) using unpaired two-way ANOVA with the Bonferroni posttest. p values are shown on the graphs. Source data are provided as a Source Data file. Fig. 6G, J Created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license https://creativecommons.org/licenses/by-nc-nd/4.0/deed.en .
Article Snippet: To analyze human ILC2s, antibodies against CD45 (clone HI30; #MHCD4512; Invitrogen; 1:500), lineage markers (CD2, clone RPA-2.10, CD3, clone OKT3, CD14, clone 61D3, CD16, clone CB16, CD19, clone HIB19, CD56, clone TULY56, and CD235a, clone HIR2; #22-7778-72; eBioscience; 1:250), CRTH2 (clone BM16; #12-2949-41; eBioscience; 1:500), CD127 (clone eBioRDR5; #15-1278-42; Invitrogen; 1:500), GATA3 (clone TWAJ; #48-9966-42; Invitrogen; 1:200), ST2 (clone hIL33Rcap; #46-9338-42; Invitrogen; 1:200), KLRG1 (clone 47-9488-41; #47-9488-41; eBioscience; 1:200), IL-17RB (clone 170220; #FAB1207A; R&D systems; 1:200), IL-13 (clone 85BRD; #51-7136-42; Invitrogen; 1:200), IL-5 (clone TRFK5; #48-7052-82; Invitrogen; 1:200), Ki-67 (clone SolA15; #53-5698-82; Invitrogen; 1:500) and corresponding isotype controls were applied.
Techniques: Isolation, Quantitative RT-PCR, Expressing, Transfection, Negative Control, Knockdown, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Injection, In Vivo, Adoptive Transfer Assay, Comparison
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![A Flow chart of asthma mice model establishment and sample collection. B , C On days 21 to 23, BrdU (0.8 mg/mice) was given intranasally. At indicated time points, single-cell suspension was prepared and subjected to flow cytometry analysis; Dynamic changes of the percentage B and numbers C of CD45 + lineage – CD90.2 + GATA3 + ST2 – KLRG1 + IL-17RB + ILC2s in siLP. D The proportions of BrdU + ST2 – KLRG1 + IL-17RB + ILC2s in siLP of HDM-challenged mice were analyzed by flow cytometry. A control histogram (shaded in blue) was generated using cells from ctrl mice stained with BrdU. E During asthma remission (AR) (day 30) of the i.p. HDM sensitization model, three ILC2 subsets including KLRG1 – , KLRG1 + IL-17RB + and KLRG1 + IL-17RB + (marked as AR-KLRG1 – , AR-KLRG1 + IL-17RB + and AR-KLRG1 + IL-17RB + ) were purified from the total CD45 + lineage – CD90.2 + ST2 – cells from siLP. Besides, ST2 – KLRG1 + IL-17RB + ILC2s (marked as naive-KLRG1 + IL-17RB + ILC2s) were sorted from siLP of untreated WT mice. Cells were then cultured (5 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\times$$\end{document} × 10 3 /well) with 10 ng/mL IL-2 and 20 ng/mL IL-7, and treated with (alarmins + ) or without (alarmins-) 200 ng/mL IL-25 and 200 ng/mL IL-33 every other day for 14 days. Subsequently, cells were collected for further analysis. (F-H) Ki-67 + percentage F , number ( G ) and fold changes H of expansion of cells were determined. I Representative plots and statistical results (bar graph) showing the percentages of IL-13 + and IL-5 + of these four ILC2 subsets after stimulation with PMA, ionomycin, brefeldin A and monensin. Data are representative of four independent experiments. Means \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\pm \,$$\end{document} ± SD; n = 5 for the naive-KLRG1 + IL-17RB + group, and n = 9 for the other groups. B , C statistical comparisons were performed using unpaired, two-sided Welch’s t test. F – I unpaired two-way ANOVA with the Bonferroni posttest analysis was performed. p values are shown on the graphs. Source data are provided as a Source Data file. Fig. 5E Created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license https://creativecommons.org/licenses/by-nc-nd/4.0/deed.en .](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_1517/pmc11381517/pmc11381517__41467_2024_52252_Fig5_HTML.jpg)
