Fig. 3. Carinh protects against DSS-induced colitis by promoting Irf1 transcription in myeloid cells.

a RNA-seq of BMDMs from Carinh^WT and Carinh^KO mice. Volcano plot showing distribution of DEGs between Carinh^WT and Carinh^KO BMDMs. Blue dots (right) and blue dots (left) correspond to genes with significantly increased or decreased expression under each condition (fold changes ratio greater than 1.5 or less than –1.5 with a P-value < 0.01). The x-axis shows the log₂ of the fold changes of expression and the y-axis shows the P-value (–log₁₀) for each gene. b Western blot assay detecting IRF1 protein expression in CD11b⁺ myeloid cells sorted from intestines of DSS-treated Carinh^WT and Carinh^KO mice (n = 3). c–e DSS-induced colitis model in LysM-cre Irf1^△Myeloid mice. Irf1^△Myeloid mice were administered with 2.5% DSS. Colitis was monitored by body weight loss (c), colon shortening (d) and H&E staining of colon tissues (e). For H&E staining (e): left, representative pictures. Scale bars, 50 μm. Right, quantification of corresponding histology scores. 5 views per mice, 6 mice per group. f–h DSS-induced colitis model in Irf1^△IEC mice. Irf1^△IEC mice were administrated with 2.5% DSS. Colitis was monitored by body weight loss (f), colon shortening (g) and colon histology scores (h). DSS-induced colitis model in Irf1^{△T cell} mice. Irf1^{△T cell} mice were administrated with 2.5% DSS. Colitis was monitored by body weight loss (i), colon shortening (j) and colon histology scores (k). qPCR analyses of Carinh and Irf1 mRNA expression in L929 cells transduced with Carinh-specific shRNAs or scramble control (l), or in murine macrophage cell lines (J774) transduced with Carinh plasmid or empty vector (m). n = 3 per group. n ChIP-qPCR for H3K27ac at the Irf1 promoter in BM cells. A–E represent selected qPCR primers in the indicated locations at Irf1 promoter; F represents qPCR primer in non-relevant region. Data are presented as enrichment fold over the IgG control. n = 3 per group. o Co-immunoprecipitation analysis between Carinh RNA and H3K27ac modifier p300/CBP. Protein extracts from HEK293 cells transduced with HA-tagged p300 or HA-tagged CBP and Carinh RNA, were immunoprecipitated with HA antibodies. RNAs binding to p300 or CBP were assayed by qPCR. P1 to P6 represent selected qPCR primers in Carinh region. U1 represents a non-relevant control. Data are presented as relative expression of input. n = 3 per group. p qPCR analyses of Il18bp expression in L929 cells transduced with Carinh-specific shRNAs or scramble control (left), or in murine macrophage cell lines (J774) transduced with Carinh plasmid or empty vector (right). n = 3 per group. q ELISA detection of IL-18BP levels in colon homogenates of DSS-induced Carinh^WT (n = 8) and Carinh^KO (n = 12) mice. IL-18BP supplement in DSS-induced colitis model. Carinh^KO mice were treated with 2.5% DSS and intraperitoneally (i.p.) injected with or without IL-18BP. Colitis was monitored by body weight loss (r), colon shortening (s) and H&E staining of colon tissues (t). For H&E staining (t): left, representative pictures. Scale bars, 50 μm. Right, quantification of corresponding histology scores. 5 views per mice, Carinh^WT mice n = 4, Carinh^KO mice n = 5, Carinh^KO mice with IL-18BP treatment n = 4. Data in d, e, g, j, s and t are representative of 3 independent experiments. Body weight in f and i are pooled from 3 independent experiments. Data in q are pooled from 2 independent experiments. Data from in vitro experiments are representative of at least three independent experiments. Data represent means ± SEM. Body weight changes in c, f, i and r were analyzed by two-way ANOVA. Histology scores (t) were analyzed by one-way ANOVA. Unpaired two-tailed Student’s t-tests were used for the other statistical analyses. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; ns, not significant.