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. Author manuscript; available in PMC: 2021 May 1.
Published in final edited form as: Nat Immunol. 2020 Aug 24;21(11):1430–1443. doi: 10.1038/s41590-020-0764-8

Extended Data Fig. 8: Rspondin3 induces TET2-mediated DNA hydroxymethylation of anti-inflammatory genes.

Extended Data Fig. 8:

(a) hMeDIP performed on WT, Lgr4Mφ−/−, Ctnnb1Mφ−/− and Tet2Mφ−/− BMDMs treated with Rspondin3, LPS alone or combination of both, and qPCR with primers targeting the proximal promoters of the indicated genes were used to detect the enrichment of 5hmC; Data are representative of three independent experiments, n=3 samples per group (mean ± sd); Statistical significance was determined by two-way ANOVA with Tukey’s multiple comparisons test using GraphPad Prism. P values (left to right) are: Chil3 (****P<0.0001, ****P<0.0001, ns P>0.9999, ns P>0.9999, ns P=0.9998, ns P>0.9999, ns P=0.9983, ns P>0.9999), Retnla (****P<0.0001, ****P<0.0001, ns P>0.9999, ns P>0.9999, P>0.9999, ns P>0.9999, ns P>0.9999, ns P>0.9999). (b) ChIP with an anti-H3K4me3 antibody were performed on WT, Lgr4Mφ−/−, Ctnnb1Mφ−/− and Tet2Mφ−/− BMDMs treated with Rspondin3, LPS alone or in combination, and qPCR with primers targeting the proximal promoters of the indicated genes were used to detect the enrichment of H3K4me3; Data are representative of three independent experiments, n=3 samples per group (mean ± sd); Statistical significance was determined by two-way ANOVA with Tukey’s multiple comparisons test using GraphPad Prism. P values (left to right) are: Chil3 (****P<0.0001, ****P<0.0001, ns P=0.9920, ns P>0.9999, ns P=0.9883, ns P=0.9843, ns P=0.6545, ns P>0.9900), Retnla (****P<0.0001, ****P<0.0001, ns P=0.9998, ns P>0.9999, P=0.9992, ns P>0.9999, ns P=0.8940, ns P=0.9994). (c) Bar figure showing levels of anti-inflammatory markers and pro-inflammatory markers in lung IM in WT and Tet2Mφ−/− mice with or without rRspondin3 i.v. under basal and post-sublethal LPS challenge for 24h as measured by CyTOF (data are representative of three independent experiments with five mice per group). Graphs show the mean ± s.d, with each dot representing an individual mouse. Statistical significance was determined by two-way ANOVA with Tukey’s multiple comparisons test using GraphPad Prism with individual P values (left to right) are: CD206 (****P<0.0001, ****P<0.0001, ns P=0.9618), CD301 (**P=0.0015, ****P<0.0001, ns P=0.9723), Arginase 1 (****P<0.0001, ****P<0.0001, ns P=0.9945), IL-10 (**P=0.0019, ****P<0.0001, ns P=0.8873), CD86 (****P<0.0001, ***P=0.0002, ns P=0.9799), CD80 (****P<0.0001, ns P=0.1255, ns P=0.8631), iNOS (****P<0.0001, ns P=0.6380, ns P=0.8866), TNF (****P<0.0001, **P=0.0012, ns P=0.8863). (d) Absolute cell number for IM in WT and Tet2Mφ−/− mice with or without rRspondin3 i.v. under basal conditions and post sublethal LPS challenge for 24h measured by CyTOF (Data are representative of three independent experiments with five mice per group). Graphs show the mean ± s.d, with each dot representing an individual mouse. Statistical significance was determined by two-way ANOVA with Tukey’s multiple comparisons test using GraphPad Prism with individual P values (left to right) are ns P>0.4182, ****P<0.0001, ****P<0.0001, ns P=0.9444; (e) Representative confocal images for immunofluorescent staining with anti-TET2 and anti-β-catenin antibodies performed on BMDMs stimulated with Rspondin3 or PBS from three independent repeats, and quantified data are shown (right) with n=5 samples per group (mean ± sd); Statistical significance was determined by two-tailed unpaired t test using GraphPad Prism. **** P<0.0001. (f) Representative confocal images for in situ proximity ligation assay (PLA) with anti-TET2 and anti-β-catenin antibodies to detect interactions were performed on BMDMs stimulated with Rspondin3 or PBS (three independent repeats), and quantified data are shown (right) with n=5 samples per group (mean ± sd); Statistical significance was determined by two-tailed unpaired t test using GraphPad Prism. **** P<0.0001. (g) Model: The angiocrine-metabolic-epigenetic axis regulates lung IM phenotypic transition. Lung ECs release Rspondin3, which binds to its cell surface receptor-LGR4 in IM and activates β-catenin leading to increased α-ketoglutarate concentration through activation of glutaminolysis, followed by induction of TET2 mediated epigenetic reprograming. TET2 mediated DNA hydroxymethylation increases expression of anti-inflammatory genes in IM and prevents inflammatory lung injury.