Fig 1: Carinh protects against DSS-induced colitis by promoting Irf1 transcription in myeloid cells.a RNA-seq of BMDMs from CarinhWT and CarinhKO mice. Volcano plot showing distribution of DEGs between CarinhWT and CarinhKO 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 CarinhWT and CarinhKO 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 CarinhWT (n = 8) and CarinhKO (n = 12) mice. IL-18BP supplement in DSS-induced colitis model. CarinhKO 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, CarinhWT mice n = 4, CarinhKO mice n = 5, CarinhKO 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.
Fig 2: A causal variant in human CARINH locus increases IBD risk by impairing the inducible expression of CARINH.a Summary of IBD-associated variants in the 95% credible set in the chr5: 131.2MB-132.2MB region. Results are taken from reference.1 Prob, the posterior probability for a variant to be causal to IBD; OR, odds ratio; SE, standard error. b Genetic associations with IBD in the CARINH region. P-values were taken from an association study of IBD.1 The region is refined as 400 kb up- and downstream of the most significantly associated variant: rs2188962. c The T allele of rs2188962 increases the genetic risk for IBD. The proportion of individuals diagnosed with IBD for each genotype (CC, CT and TT) of rs2188962 from the International Inflammatory Bowel Disease Genetics Consortium data (35,109 IBD patients and 35,761 controls) were calculated to evaluate the relative risk to IBD. The relative risk was calculated as the ratio of the proportions using genotype CC as the baseline genotype. Error bar indicates 95% confidence interval. d qPCR analysis of human CARINH, IRF1 and IL18BP mRNA expression in CRISPR-edited HeLa cell clones with rs2188962 (C/T) variant of each genotype (CC, CT and TT) treated with or without microbe component mimic (Poly I:C). n = 3 per group. NT, no treatment. The data in d are representative of at least 3 independent experiments. Data represent means ± SEM.
Fig 3: CARINH is functionally conserved as a regulator of inflammation in human.a CARINH RNA expression in human gut specimens from control individuals (left, n = 10), UC patients (middle, n = 10), and CD patients (right, n = 10), assessed by RNA FISH. Representative pictures are shown. Scale bar, 50 μm. b Quantification of the FISH results from a, shown as the CARINH RNA-positive cell count per mm2. n = 10 per group. c CARINH and IRF1 mRNA levels are detected by qPCR in terminal ileum biopsy specimens. Control, n = 11; CD, n = 19. d CARINH and IRF1 mRNA levels are detected by qPCR in PBMCs of IBD patients. The correlation was analyzed between CARINH RNA and IRF1 mRNA levels. e FISH and immunofluorescence staining, showing the co-localization of CARINH RNA (red) with CD11b (green). Yellow indicates co-localization. DAPI (blue) stains cell nuclei. Scale bars, 20 μm. f Immunofluorescence staining showing the co-localization of IRF1 (green) with CD11b (red). Yellow indicates co-localization. DAPI (blue) stains cell nuclei. Scale bars, 20 μm. g FISH and immunofluorescent staining showing the co-localization of CARINH RNA (red) with IRF1 (green). Yellow indicates co-localization. DAPI (blue) stains the nuclear of cell. The bottom panel of images are magnified views of the areas indicated by white line boxes above. Up panel scale bar, 50 μm. Bottom panel scale bar, 20 μm. h, i qPCR analyses of human CARINH, IRF1 and IL18BP mRNA expression in THP-1 cells transduced with human CARINH-specific siRNAs or scramble control (h), and with human CARINH-expressing vector or empty vector (i). n = 3 per group. j, k qPCR analyses of human IRF1, CARINH and IL18BP mRNA expression in THP-1 cells transduced with human IRF1-specific siRNAs or scramble control (j), and with human IRF1-expressing vector or empty vector (k). n = 3 per group. The data in h–k are representative of at least three independent experiments. Data represent means ± SEM. Data in b and h were analyzed by one-way ANOVA. Unpaired two-tailed Student’s t-tests were used for other statistical analyses. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
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