Extended Data Fig. 7: Increased SDHA levels reduce T cell mediated intestinal damage.

(a, b) Immunoblot and protein density quantification of SDHA (a) and succinate levels (b) in colonic IECs from naive C57BL/6 and Irg1^-/-mice (n = 4). (c-f) C57BL/6 animals Irg1^-/- animals received HCT (BALB/c → C57BL/6 or Irg1^-/-) (c) Images (left) and fluorescent intensity (right) of immunofluorescence staining of colon on 21 days after post allo-HCT (Complex IV = green, SDHA = red, DAPI = blue, n = 3). (d) Succinate levels in colon IECs at day21 post allo-HCT (n = 3). Clinical GVHD severity (e) and pathological GVHD score (f) of ileum and colon after post HCT (Clinical GVHD severity, Syn C57BL/6: n = 8, Allo C57BL/6 and Irg1^-/-: n = 15, pathological GVHD score, n = 3). (g) Colonic IECs from C57BL/6, Sdha^∆/IEC and Irg1^-/- were used as target cells for CTL assay (2 hr co-culture with effector T cells). Cell death was analyzed (n = 3). (h, i) PCECs mediated by CRISPR-Cas9 targeting IRG1 were treated with LPS (100 ng/ml)/ IFNγ(10 ng/ml) for 6 hr. (h) Irg1 gene expression levels (normalized to Gapdh expression, n = 4 cells examined over 3 independent experiments). (i) Control and IRG1 KO PCECs were used as target cells for CTL assay (7 hr co-culture with effector T cells). Data are from 1 of 3 similar experiments) (j, k) C57BL/6 animals received HCT (BALB/c → C57BL/6) and treated with vehicle or butyrate. (j) Representative images (left) of immunofluorescence staining (Complex IV = green, SDHA = red, DAPI = blue) (left) and fluorescent intensity (right) of SDHA in colon on day21 post allo-HCT (n = 5). (k) Succinate levels in IECs from colon and ileum on day7 (top) and day21 (bottom) post allo-HCT (n = 5). (l) ChIP assay of 1 mM butyrate-treated isolated colonic IECs from naive C57BL/6 binding control IgG, pol II or acetylated histone H4 in the promoter regions of Sdha and Sdhb (n = 5). Data (a-d, f, g, j-l) are from biologically independent animals. Scale bar (c, j) = 50 µm. All statistical analysis by two-tailed Mann-Whitney test (a, h, j), two-tailed unpaired t-test (b, d, f, i, k, l), Kruskal-Wallis analysis with Dunn’s post hoc test (e) or ANOVA analysis with Tukey post hoc test (g) (mean ± s.e.m.).