Loss of Runx3 function in leukocytes is associated with spontaneously developed colitis and gastric mucosal hyperplasia

Abstract

RUNX transcription factors are key regulators of lineage-specific gene expression and might be involved in autoimmune diseases. Runx3 plays a role during the development of sensory neurons and T cells and regulates transforming growth factor beta (TGF-beta) signaling in dendritic cells. Here, we report that at 4 weeks of age, Runx3 knockout (KO) mice spontaneously develop inflammatory bowel disease (IBD) characterized by leukocyte infiltration, mucosal hyperplasia, formation of lymphoid clusters, and increased production of IgA. Additionally, at a considerably older age (8 months), the KO mice also develop progressive hyperplasia of the gastric mucosa associated with disturbed epithelial differentiation and cellular hyaline degeneration. Analysis of cytokines in the colonic mucosa of Runx3 KO mice revealed a mixed T helper 1/T helper 2 response. By using immunohistochemistry and RNA in situ hybridization, Runx3 expression in the gastrointestinal tract is detected in lymphoid and myeloid populations but not in the epithelium. The data indicate that loss of leukocytic cell-autonomous function of Runx3 results in IBD and gastric lesion in the KO mice. IBD in humans is viewed as a complex genetic disorder. Several susceptibility loci were identified on different human chromosomes including the chromosomal region 1p36 where RUNX3 resides. It is thus tempting to speculate that mutations in RUNX3 may constitute an IBD risk factor in humans.

Fig. 1.

Pathologic findings in the large intestines of Runx3 KO mice in comparison with WT mice. (A and B) Gross specimens of the descending colon and rectum show marked thickening of the intestinal wall and absence of fecal pellets in the KO. (C and D) H&E-stained sections of the colon show severe mucosal and submucosal thickening in KO due to mixed inflammatory cellular infiltration with crypt hyperplasia and loss. (E and F) Masson's trichrome stain demonstrates abnormal deposition of collagen (arrows in F), stained blue, in the KO lamina propria. (G and H) Immunostaining with anti-Ki-67, which directly monitors cell divisions, shows an increase in proliferating cells (stained brown) in crypts of the KO cecum. (Magnification: C–F, ×10; G and H, ×20.)

Fig. 2.

Characterization of leukocyte infiltrates in the inflamed Runx3 KO colon. IHC on WT and KO colon sections (×4) (A) and on a KO B cell cluster (×10) (B) with anti-CD45R for detection of B cells, anti-IgA for detection of IgA, anti-GFP for detection of DC (13), anti-CD3 for detection of T cells, and anti-F4/80 for detection of macrophages. (Scale bars: A, 250 μm; B, 50 μm.)

Fig. 3.

Pathologic findings in the stomach of the Runx3 KO. (A and B) Gross specimens of hemisected stomachs show contraction and marked thickening of the entire glandular portion in the KO (B), compared with WT (A). e, Esophagus; f, forestomach; g, glandular stomach; and p, pylorus. (C and D) H&E-stained sections of the gastric wall in the fundic region. There is prominent mucosal hyperplasia and hyalinosis in the KO. tm, Tunica muscularis. (E) IHC with anti-YM1/2 protein demonstrates positive globular material within the cytoplasm of tall columnar epithelial cells. (F) High magnification of the area indicated in D by a square shows accumulation of eosinophilic rectangular crystalline material within the lumen of hyperplastic glands. (G) Cystic dilation and collapse of abnormal mucosa into subjacent layers, or “mucosal herniation.” Asterisks denote the muscularis mucosa below, in which five dilated glands are seen, two of which are indicated by h. The four glands on the left are above the tunica muscularis (tm). One gland reaches but does not invade the serosa (in the area between the two arrows). Inset shows the epithelial lining of the cysts (area of Inset indicated by a square in the main photo). (H) A focus of mucosal dysplasia. In the gland at the center of the field, there is cellular atypia in the form of loss of orderly stratification, nuclear crowding, and increased nuclear/cytoplasmic ratio. Relatively well differentiated glands are seen at the bottom right and left-hand side of the field. (Magnification: C and D, ×10; E, ×40; F, ×20; G, ×2; Inset, ×20; and H, ×20.)

Fig. 4.

Expression of Th1 and Th2 cytokines in inflamed Runx3 KO colon. RT-PCR analysis of cytokines and transcription factors (TF), in colon RNA of young (9.5 weeks) and adult (9 months) WT and KO mice. Reg, regulatory cytokines. A detailed account of primers used for PCR is available in Supporting Text. Depicted are representative examples of several independent repetitions.

Fig. 5.

Expression of Runx3 and Runx1 in GIT and DRG of WT mice. (A) Expression of Runx3 in adult GIT (stomach, small intestine, and large intestine). (B) Expression of Runx3 and Runx1 in embryonic day 16.5 embryos. Shown are RISH analysis of DRG (Top) and stomach (Middle) and IHC analysis of stomach (Bottom). F, forestomach; G, glandular stomach.