Volume 125, Issue 5, Pages 1388-1397 (November 2003) Bacterial colonization leads to the colonic secretion of RELMβ/FIZZ2, a novel goblet cell-specific protein  Weimian He, Mei-Lun Wang, Han-Qing Jiang, Claire M Steppan, Marcus E Shin, M.Christine Thurnheer, John J Cebra, Mitchell A Lazar, Gary D Wu  Gastroenterology  Volume 125, Issue 5, Pages 1388-1397 (November 2003) DOI: 10.1016/j.gastro.2003.07.009

Figure 1 Immunolocalization of RELMβ protein in the colon. (A) Immunohistochemistry of RELMβ in a Swiss roll of mouse colon. (B) Coincubation of the RELMβ antibody and the immunogen peptide eliminates the RELMβ signal. (C) High-power magnification of distal mouse colon stained for RELMβ (arrow, release of RELMβ containing mucin granule into the lumen of the colon). (Original magnification: A and B, 40×; C, 400×.) Gastroenterology 2003 125, 1388-1397DOI: (10.1016/j.gastro.2003.07.009)

Figure 2 RELMβ expression, which is absent in the distal colon of germ-free animals, can be induced by bacterial colonization. (A) Swiss-rolled colon from a germ-free mouse stained for RELMβ. (B) High-power magnification of the distal colon of a germ-free mouse stained for RELMβ. (C) Cecum of a conventionally housed mouse stained for RELMβ (arrows indicate RELMβ-containing goblet cells on the surface epithelium). (D) Cecum of a germ-free mouse stained for RELMβ. (E) Swiss-rolled colon stained for RELMβ from a former germ-free mouse conventionalized in a SPF environment for 2 weeks. (F) Cecum of a conventionalized mouse stained for RELMβ. (G) Immunoblot for RELMβ using protein isolated from the stool of a germ-free BALB/c and C.B17.SCID mouse after introduction into a conventional environment for the time periods indicated. The results shown are representative of 4 independent BALB/c and C.B17.SCID animals. DC, distal colon in the center; PC, proximal colon in the periphery; Con, proteins isolated from the stool of a conventionally housed mouse. (Original magnification: A, 20×; B, 200×; C and D, 100×; E and F, 40×.) Gastroenterology 2003 125, 1388-1397DOI: (10.1016/j.gastro.2003.07.009)

Figure 3 RELMβ is secreted into the stool, where it exists as a homodimer under nonreducing conditions. (A) RELMβ isolated from stool of 2 mice was electrophoresed under both reducing and nonreducing conditions and detected by immunoblot. (B) Immunoblot of RELMβ in protein isolated from 3 human stool samples. (C) Northern blot for RELMβ using RNA isolated from 3 different colon cancer cell lines. Flg, flagged tagged recombinant RELMβ; EtBr, ethidium bromide-stained gel of RNA before transfer. Gastroenterology 2003 125, 1388-1397DOI: (10.1016/j.gastro.2003.07.009)

Figure 4 RELMβ expression is reduced in germ-free animals. (A) Northern blot of RNA isolated from several conventionally housed and germ-free mice. The same blot was probed for RELMβ, DRA, c-myc, and intestinal trefoil factor (TFF3). (B) Immunoblot for RELMβ using protein isolated from the colon of 3 conventionally housed and germ-free mice. Flg, flagged tagged recombinant RELMβ. (C) Immunoblot for RELMβ using protein isolated from the stool of 5 conventionally housed and germ-free mice. Gastroenterology 2003 125, 1388-1397DOI: (10.1016/j.gastro.2003.07.009)

Figure 5 High levels of RELMβ mRNA and stool protein expression are observed specifically in conventionally housed mice. (Upper panel) Quantification of RELMβ, Muc2, and TFF3 mRNA expression by kinetic RT-PCR using RNA isolated from the colon of germ-free (GF), conventionally housed (Con), and gnotobiotic mice colonized with either segmented filamentous bacteria (SFB), H. muridarum (HM), Schaedler’s E. coli (EC), or altered Schaedler flora (ASF). Data, normalized to glyceraldehyde-3-phosphate dehydrogenase, are expressed as the fold induction relative to that observed in germ-free mice (arbitrarily set at a value of 1). n = 3, mean ± SD. (Middle panel) Immunoblot for RELMβ using protein isolated from the stool of mice housed in a germ-free or conventional environment as well as those colonized with the commensal bacteria indicated. (Bottom panel) Gold stain of the immunoblot shown in the middle panel to demonstrate equivalent loading of stool proteins. Gastroenterology 2003 125, 1388-1397DOI: (10.1016/j.gastro.2003.07.009)

Figure 6 RELMβ mRNA expression can be induced by either LPS or IL-13 in intestinal cell lines. (A) Diagram of the human RELMβ promoter showing cis-acting sequences for transcription factors shown to regulate the transcription of goblet cell as well as inflammatory response genes. (B) Quantitative RT-PCR for RELMβ using RNA isolated from intestinal cell lines stimulated for 24 hours with LPS using the indicated concentrations. (C) Quantitative RT-PCR for RELMβ using RNA isolated from LS174T cells stimulated with IL-13 (10 ng/mL) for the times indicated. Gastroenterology 2003 125, 1388-1397DOI: (10.1016/j.gastro.2003.07.009)