Altered expression of Butyrophilin (BTN) and BTN-like (BTNL) genes in intestinal inflammation and colon cancer

Abstract

Several Butyrophilin (BTN) and Btn-like (BTNL) molecules control T lymphocyte responses, and are genetically associated with inflammatory disorders and cancer. In this study, we present a comprehensive expression analysis of human and murine BTN and BTNL genes in conditions associated with intestinal inflammation and cancer. Using real-time PCR, expression of human BTN and BTNL genes was analyzed in samples from patients with ulcerative colitis, irritable bowel syndrome, and colon tumors. Expression of murine Btn and Btnl genes was examined in mouse models of spontaneous colitis (Muc2^-/-) and intestinal tumorigenesis (Apc^Min/+). Our analysis indicates a strong association of several of the human genes with ulcerative colitis and colon cancer; while especially BTN1A1, BTN2A2, BTN3A3, and BTNL8 were significantly altered in inflammation, colonic tumors exhibited significantly decreased levels of BTNL2, BTNL3, BTNL8, and BTNL9 as compared to unaffected tissue. Colonic inflammation in Muc2^-/- mice significantly down-regulated the expression of particularly Btnl1, Btnl4, and Btnl6 mRNA, and intestinal polyps derived from Apc^Min/+ mice displayed altered levels of Btn1a1, Btn2a2, and Btnl1 transcripts. Thus, our data present an association of BTN and BTNL genes with intestinal inflammation and cancer and represent a valuable resource for further studies of this gene family.

Keywords: Butyrophilin (Btn)‐like (Btnl); colon cancer; immune regulation; intestinal inflammation; irritable bowel syndrome (IBS); ulcerative colitis (UC).

Figure 1

Expression of BTN and BTNL genes in human (A) and mouse (B) colon. Real‐time PCR analysis was conducted in colon biopsies of healthy subjects (n = 18) (A), and colonic tissue from C57BL/6 mice (n = 10) (B). The expression of Btn1a1, Btn2a2, and Btnl9 was below the limit of detection for some of the animals. Each qPCR analysis was run in duplicate. Results were analyzed using the 2^−ΔCt method with HPRT1 (A) and β‐actin (B) as a reference gene. Symbols represent individual values and horizontal lines the median.

Figure 2

Expression of human BTN and BTNL genes in intestinal inflammation and colon cancer. (A) Gene expression in total RNA derived from colon of healthy individuals (n = 18), patients with IBS (n = 8) and UC (n = 16) was assessed by qPCR, run in duplicates, and determined using the 2^−ΔCt method with HPRT1 as a reference gene. Statistical significance between groups was assessed using Kruskal–Wallis test followed by Dunn's multiple comparison test (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001). Symbols represent individual values and horizontal lines the median. (B) Gene expression in tumor tissue and unaffected tissue from colon cancer patients (n = 17) was analyzed by qPCR, run in duplicates, and determined using the 2^−ΔCt method with HPRT1 as a reference gene. Wilcoxon matched‐pairs signed rank test was used for statistical analysis (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001). Connecting lines show values from samples taken from the same individual.

Figure 3

Expression of murine Btn and Btnl genes in intestinal inflammation and cancer. (A) Expression of Btn and Btnl genes in proximal, middle and distal colon sections of Muc2 ^+/− (n = 10) and Muc2 ^−/− (n = 10) mice was analyzed by qPCR, run in duplicates, and results were normalized to β‐actin. The expression of Btn1a1, Btn2a2, and Btnl9 was below the limit of detection for some of the animals. Statistical significance was assessed using the Mann–Whitney test (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001). Symbols represent individual values and horizontal lines the median. (B) Expression of Btn and Btnl genes was examined in intestinal polyps (TUM) and adjacent unaffected intestinal tissue of Apc ^Min/+ mice (n = 10) by qPCR, run in duplicates, and normalized against β‐actin. Wilcoxon matched‐pairs signed rank test was used for statistical analysis (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001). Connecting lines show values from samples taken from the same mouse.