Extended Data Fig. 6: Chemical inhibition and genetic ablation of SDHA in IECs regulate the severity of GVHD.

(a) Representative images of SDH enzyme staining of colon from naive C57BL/6 12 hours after vehicle, malonate (5 g kg^-1) or itaconate (2.5 g kg^-1) treatment. (Scale bar 100 µm, n = 2). (b-e) C57BL/6 or Sdhaf1^-/- animals received 10 Gy total body irradiation followed by 3×10⁶T cells along with 5×10⁶ TCD-BM cells from either syngeneic C57BL/6 or allogeneic BALB/c donors. (b) Survival of C57BL/6 recipients treated with vehicle or atpeninA5 (9 µg kg^-1) every other day post HCT (n = 8). (c) C57BL/6 recipients treated with vehicle, malonate (5 g kg^-1) or itaconate (2.5 g kg^-1) every other day post HCT. Pathological GVHD scores in liver, skin and lung 7days post HCT (n = 6). (d) Succinate levels in isolated IECs from colon of naive C57BL/6 mice and Sdhaf1^-/- mice (n = 4). (e) Pathological GVHD scores in liver, skin and lung 7days post HCT from C57BL/6 and Sdhaf1^-/- recipients (n = 6). (f) C57BL/6 and Sdhaf1^-/- animals were treated with 2.5 % DSS in drinking water for 7 days. Time course of body weight changes after DSS administration (n = 6). (g) Scheme illustrating the strategy used to generate Sdha floxed and excised alleles in IECs. (h) Expression of Sdha mRNA in CD326⁺ isolated IECs from six-week-old Sdha^∆/IEC relative to Sdha^fl/IEC mice (n = 4). Sdha expression was normalized to Gapdh expression. (i) Immunoblot of SDHA and TOM-20 in mitochondria of IECs from six-week-old Sdha^∆/IEC and Sdha^fl/IEC mice. (j) Representative images of immunofluorescence staining of colon, ileum, liver, skin and heart from six-week-old Sdha^∆/IEC and Sdha^fl/IEC mice (Complex IV = green, SDHA = red, DAPI = blue, scale bar=50 µm, n = 4). Data (a-f, h) are from biologically independent animals. All statistical analysis by log-rank test (b), Kruskal-Wallis analysis with Dunn’s post hoc test (c), two-tailed unpaired t-test (d), or two-tailed Mann-Whitney test (e, h) (mean ± s.e.m.).

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