Fig 2: Knockout of OTUD4 in IECs leads tohyper-resistancetoS.t. infection.(A) Survival of Otud4fl/fl (n = 7) and Vil-Cre;Otud4fl/fl (n = 9) mice that were injected with Salmonella typhimurium (S.t.) (2 × 107 c.f.u. per mouse) by gavage.(B) Body weight change of Otud4fl/fl (n = 12) and Vil-Cre;Otud4fl/fl (n = 12) mice treated as in (A).(C) A representative image and the lengths of colons of Otud4fl/fl (n = 12) and Vil-Cre;Otud4fl/fl (n = 12) mice treated as in (A) and sacrificed at day 6.(D) Representative images of HE-stained colon sections of Otud4fl/fl and Vil-Cre;Otud4fl/fl mice treated as in (A) and sacrificed at day 6.(E) Bacterial counts (CFU/g) of feces, cecum, liver, and spleen from Otud4fl/fl (n = 5) and Vil-Cre;Otud4fl/fl (n = 5) mice treated as in (A).(F) qRT-PCR analysis of expression levels of the indicated genes of IECs from Otud4fl/fl (n = 6) and Vil-Cre;Otud4fl/fl (n = 6) mice at day 2 after injection of S.t. (2 × 107 c.f.u. per mouse) by gavage.∗P ​< ​0.05, ∗∗P ​< ​0.01, ∗∗∗P < 0.001 (Log-rank test in A, Student's unpaired t-test in B, C, E, F). Graphs show mean ± S.D. (B, C, E, F). Red and black scale bars represent 2 cm and 400 μm, respectively (C, D). Data are representative of two independent experiments (A, D–F) or combined two independent experiments (B, C).

Fig 3: OTUD4 inhibits the expression of AMPs by targeting MyD88.(A) Whole-cell extract of IECs from wild-type C57BL/6 mice was immunoprecipitated with control IgG or anti-OTUD4 and subjected to immunoblot analysis.(B) Pulldown (with GST beads and GST-K63-Ub-TUBE) and immunoblot analysis of IECs from Otud4fl/fl (n = 2) and Vil-Cre;Otud4fl/fl (n = 2) mice that were uninduced, given 2.5% DSS in drinking water for 2 days or infected with S.t. (2 × 107 c.f.u. per mouse) by gavage for 2 days. TUBE, tandem ubiquitin binding entity.(C) Immunoblot analysis of the indicated proteins in IECs from Otud4fl/fl (n = 2) and Vil-Cre;Otud4fl/fl (n = 2) mice treated as in (B).(D) qRT-PCR analysis of the indicated genes of intestinal organoids from Otud4fl/fl and Vil-Cre;Otud4fl/fl mice that were unstimulated or stimulated with LPS (10 µg/mL) or PGN (10 µg/mL) in the presence or absence of ST2825 (a MyD88 inhibitor, 10 µM) for 4 h.(E) qRT-PCR analysis of the indicated genes of intestinal organoids from Otud4fl/fl and Vil-Cre;Otud4fl/fl mice that were uninfected and infected with S.t. (MOI, 1:200) in the presence or absence of ST2825 (10 µM) for 2 h*P ?< ?0.05, **P ?< ?0.01, ***P < 0.001 (Student's unpaired t-test in D, E). Graphs show mean ± S.D. (D, E). Data are representative of two independent experiments (A, C, D, E) or three independent experiments (B).

Fig 4: OTUD4 in Paneth cells supportsDSS-induced colitis andS.t. infection.(A) A scheme of DSS-induced colitis (upper scheme) and body weight change (lower graph) of Otud4fl/fl (n = 7) and Def-Cre;Otud4fl/fl (n = 7) mice with 2.5% DSS for 5 days followed by normal sterile water for 2 days.(B) A representative image and the lengths of colons of Otud4fl/fl (n = 7) and Def-Cre;Otud4fl/fl (n = 7) mice treated as in (A).(C) Representative images of HE-stained colon sections of Otud4fl/fl and Def-Cre;Otud4fl/fl mice treated as in (A).(D) The pathology scores of Otud4fl/fl (n = 7) and Def-Cre;Otud4fl/fl (n = 7) mice treated as in (A).(E) qRT-PCR analysis of expression levels of the indicated genes of IECs from Otud4fl/fl (n = 6) and Def-Cre;Otud4fl/fl (n = 6) mice that were given 2.5% DSS in drinking water for 2 d.(F) Survival of Otud4fl/fl (n = 12) and Def-Cre;Otud4fl/fl (n = 8) mice that were injected with S.t. (2 × 107 c.f.u. per mouse) by gavage.(G) Body weight change of Otud4fl/fl (n = 12) and Def-Cre;Otud4fl/fl (n = 12) mice treated as in (F) and sacrificed at day 6.(H) A representative image and the lengths of colons of Otud4fl/fl (n = 12) and Def-Cre;Otud4fl/fl (n = 12) mice treated as in (F) and sacrificed at day 6.(I) Representative images of HE-stained colon sections of Otud4fl/fl and Def-Cre;Otud4fl/fl mice treated as in (F) and sacrificed at day 6.(J) Bacterial counts (CFU/g) of feces, cecum, liver, and spleen from Otud4fl/fl (n = 6) and Def-Cre;Otud4fl/fl (n = 6) mice treated as in (F).(K) qRT-PCR analysis of Def5a or Reg3g of IECs from Otud4fl/fl (n = 6) and Def-Cre;Otud4fl/fl (n = 6) mice at day 2 after injection of S.t. (2 × 107 c.f.u. per mouse) by gavage.*P ?< ?0.05, **P ?< ?0.01, ***P < 0.001 (Student's unpaired t-test in A, B, D, E, G, H, J, K, Log-rank test in F). Graphs show mean ± S.D. (A, B, D, E, G, H, J, K). Red and black scale bars represent 2 cm and 400 µm (B, C, H, I). Data are combined two independent experiments (A, B, D, F–H) or representative of two independent experiments (C, E, I, J, K).

Fig 5: Otud4 deficiency in IECs leads to increased resistance toDSS-inducedcolitis.(A) OTUD4 mRNA expression levels in colonic mucosa tissues from ulcerative colitis (UC) patients and healthy individuals (data from GEO database GDS3119) (n = 26).(B) A scheme of DSS-induced colitis (upper scheme) and body weight change (lower graph) of Otud4fl/fl (n = 14) and Vil-Cre;Otud4fl/fl (n = 14) mice treated with 2.5% DSS for 5 days followed by normal sterile water for 2 days.(C) A representative image and the lengths of colons of Otud4fl/fl (n = 14) and Vil-Cre;Otud4fl/fl (n = 14) mice treated as in (B).(D) Representative images of HE-stained colon sections of Otud4fl/fl and Vil-Cre;Otud4fl/fl mice treated as in (B).(E) The pathology scores of Otud4fl/fl (n = 14) and Vil-Cre;Otud4fl/fl (n = 14) mice treated as in (B).*P ?< ?0.05, **P ?< ?0.01, ***P < 0.001 (Student's unpaired t-test in A–C, E). Graphs show mean ± S.D. (A–C, E). Red and black scale bars represent 2 cm and 400 µm, respectively (C, D). Data are combined three independent experiments (B, C, E) or representative of three independent experiments (D).