Resistin-like molecule β regulates innate colonic function: Barrier integrity and inflammation susceptibility

Generation of RELM-β^−/− mice. RELM-β^−/− mice were developed by using VelociGene technology.¹⁴ A, Diagram shows the WT murine RELM-β gene locus and the gene-targeted locus. The RELM-β gene was replaced by a reporter-selection cassette, which consists of a β-galactosidase enzyme gene (LacZ) and a neomycin resistance gene (Neo). B, Northern blot analysis of total jejunum and colon RNA (20 μg) was used to examine RELM-β mRNA expression in WT and RELM-β^−/− mice. As an RNA loading control, the position of the 18S and 28S RNA band in the ethidium bromide (EtBr)-stained gels is also shown. Each lane represents a separate animal. C, Immunohistochemically stained sections of colon from WT and RELM-β^−/− mice using the RELM-β–specific polyclonal antiserum. Filled arrows depict RELM-β⁺ cells. Original magnification: C, ×200.

RELM-β deficiency alters transepithelial barrier permeability. A, Transepithelial resistance of colonic segments of muscle-free intestinal mucosa from WT and RELM-β^−/− mice. B, FITC-dextran (molecular weight 4400) permeability in muscle-free intestinal mucosal specimens from the colon from WT and RELM-β^−/− mice. Values are presented as means ± SEM (n = 3-8 mice per group). Statistical significance of differences was determined by using an unpaired Student t test. ^∗P < .05 compared with WT mice.

DSS treatment induces experimental colitis in WT and RELM-β^−/− mice. A, Northern blot analysis of RELM-β mRNA expression in the colons of control (top panel) and DSS-treated (middle panel) WT mice and kinetics (days 0-8) of RELM-β mRNA expression in the colons of WT mice and IL-13^−/− mice (bottom panel) on day 7. Total RNA is shown by ethidium bromide (EtBr) staining. DAI (B), weight change (C), diarrhea–rectal bleeding score (D), and colon length (E; day 7) of control- and DSS-treated WT and RELM-β^−/− mice. F, The percentage survival during the course of DSS treatment in WT and RELM-β^−/− mice. Data in Fig 3, B through F, represent the mean ± SEM of 4 to 5 mice per group from triplicate experiments. Statistical significance of differences was determined by using the Kruskal-Wallis test (^∗P < .05).

Histopathology and colonoscopy in the colons of DSS-treated WT and RELM-β^−/− mice. A-F, Representative photomicrographs of colons from control and DSS-treated WT and RELM-β^−/− mice. G-L, Representative colonoscopy photographs of colons of control and DSS-treated WT and RELM-β^−/− mice. M and N, Representative photomicrographs of immunohistochemically stained colon sections from DSS-treated WT mice using the RELM-β–specific polyclonal antiserum. Black arrows depict RELM-⁺ inflammatory cells. Black arrows in Fig 4, B, depict ulceration of the epithelial cell layer: black arrows in Fig 4, G and L, depict normal colonic vasculature (b.v., blood vessel); black arrows in Fig 4, I, H, and J, depict mucus and diarrhea (d), friable mucosa (f. m.), and rectal bleeding (b). Fig 4, H, Narrowing of colon, with evidence of a stricture. Original magnification: A-D, ×50; E and M, ×100; F and N, ×200.

Colon histopathology and colonoscopy in TNBS-treated WT and RELM-β^−/− mice. A, Percentage weight change of control-treated and TNBS-treated WT and RELM-β^−/− mice. B, Percentage survival during the course of DSS treatment in WT and RELM-β^−/− mice. C, Northern blot analysis of RELM-β mRNA expression in the colons of control-treated and TNBS-treated WT and RELM-β^−/− mice on day 5. Total RNA is shown by means of ethidium bromide (EtBr) staining. D, Hematoxylin and eosin staining of representative colon transverse sections on day 5 after TNBS administration. E, Representative colonoscopy from TNBS-treated WT and RELM-β^−/− mice. The black arrows in Fig 5, D, depict cellular infiltrate. The open arrow in Fig 5, E, depicts normal colonic vasculature, and the black arrow depicts a colonic ulcer. Data in Fig 5, A and B, represent the mean ± SEM of 4 to 5 mice per group from triplicate experiments. Statistical significance of differences was determined by using the Kruskal-Wallis test. ^∗P < .05.

Gene profile analysis of the colon in the absence of RELM-β. A, Microarray analysis of the transcripts expressed in colonic samples. RNA from each mouse was subjected to chip analysis with Affymetrix mouse Genome MOE430_2 GeneChips. The RELM-β^−/− group is composed of 5 mice, and the WT group is composed of 6 mice. The 32 genes differentially expressed (2-fold cut off and P < .05) in the RELM-β^−/− mice compared with the WT mice have been ordered (fold change); upregulated genes are shown in red, and downregulated genes are shown in blue. The magnitude of the gene changes is proportional to the darkness of the color. Each column represents an individual mouse, and each line represents a separate gene. B, Quantitative analysis of REG3β and REG3γ expression levels in the colons of WT and RELM-β^−/− mice using Affymetrix mouse Genome U430 Plus 2.0 GeneChips. C and D, Quantitative analysis of REG3β (Fig 6, C) and REG3γ (Fig 6, D) mRNA levels in the colons of WT and RELM-β^−/− mice by using real-time PCR analysis. REG3 expression was normalized to GADPH expression in each individual sample. Results are expressed as fold change over WT mice. The black line represents the mean value in each group.