The olfactory G protein-coupled receptor (Olfr-78/OR51E2) modulates the intestinal response to colitis

Abstract

Olfactory receptor-78 (Olfr-78) is a recently identified G protein-coupled receptor activated by short-chain fatty acids acetate and propionate. A suggested role for this receptor exists in the prostate where it may influence chronic inflammatory response leading to intraepithelial neoplasia. Olfr-78 has also been shown to be expressed in mouse colon. Short-chain fatty acids and their receptors are well known to modulate inflammation in the gut. Considering this possibility, we first explored if colitis regulated Olfr-78 expression in the gut, where we observed a significant reduction in the expression of Olfr-78 transcript in mouse models of dextran sodium sulfate (DSS)- and 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis. To more directly test this, mice deficient in Olfr-78 were administered with DSS in water for 7 days and were found to have increased expression of IL-1β and inflammatory signs in colon compared with control mice. Next, we explored the expression of its human counterpart olfactory receptor family 51, subfamily E, member 2 (OR51E2) in human intestinal samples and observed that it was in fact also expressed in human colon samples. RNA sequence analysis revealed significant changes in the genes involved in infection, immunity, inflammation, and colorectal cancer between wild-type and Olfr-78 knockout mice. Collectively, our findings show that Olfr-78 is highly expressed in colon and downregulated in DSS- and TNBS-induced colitis, and DSS-treated Olfr-78 null mice had increased colonic expression of cytokine RNA levels, suggesting a potential role for this receptor in intestinal inflammation. Future investigations are needed to understand how Olfr-78/OR51E2 in both mouse and human intestine modulates gastrointestinal pathophysiology.

Keywords: Olfr-78; inflammation; intestine.

Fig. 1.

Olfactory receptor-78 (Olfr-78)/olfactory receptor family 51, subfamily E, member 2 (OR51E2) expression in mouse and human intestinal samples and colon cancer cell lines. A: total RNA isolated from mucosal scrapings of different regions of mouse intestine was analyzed for Olfr-78 expression by real-time RT-PCR and normalized with GAPDH mRNA (n = 3; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ^$$P < 0.01, and ^$$$$P = 0.0001). B: RNA from different regions of human intestine was extracted and analyzed for OR51E2 expression by real-time RT-PCR and normalized with GAPDH RNA. C: RNA from Caco-2, T-84, and HT-29 colonic epithelial cells was isolated and subjected to real-time RT-PCR using OR51E2-specific primers and normalized with GAPDH mRNA.

Fig. 2.

Olfactory receptor-78 (Olfr-78) expression in colitis models. A: colonic Olfr-78 expression is reduced in mouse models of 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis, where real-time RT-PCR was performed on total RNA extracted from mucosal scrapings of distal colons from control and TNBS-treated mice using Olfr-78-specific primers and normalized with GAPDH RNA (n = 4, **P < 0.01). B: colonic Olfr-78 expression is reduced in mouse model of dextran sodium sulfate (DSS)-induced colitis, where real-time RT-PCR was performed on total RNA extracted from mucosal scrapings of distal colons from control and DSS-treated mice using Olfr-78 gene-specific primers and normalized with GAPDH mRNA (n = 4, **P < 0.01). C and D: G protein-coupled receptor (GPR) 43/41 expression is not altered in TNBS- and DSS-induced mouse colitis models. Real-time RT-PCR was performed on RNA isolated from proximal and distal colons of control and DSS- and TNBS-treated mice using GPR-43/41 gene-specific and GAPDH primers (n = 4, *P < 0.05).

Fig. 3.

A: increased expression of IL-1β in colons of olfactory receptor-78 (Olfr-78) knockout (KO) mice treated with dextran sodium sulfate (DSS). Wild-type (WT) and Olfr-78 KO male mice were administered 3% DSS in water for 7 days and RNA from distal colon was isolated for the analysis of proinflammatory cytokine IL-1β expression by real-time RT-PCR (n = 5 for WT and 7 for Olfr-78 KO; *P < 0.05). B: increased myeloperoxidase (MPO) activity in Olfr-78 KO mice treated with DSS compared with WT mice treated with DSS.

Fig. 4.

Structural changes in colons of wild-type (WT) and olfactory receptor-78 (Olfr-78) KO mice treated with dextran sodium sulfate (DSS). A: formalin-fixed distal colon tissues from WT and Olfr-78 knockout (KO) mice treated with DSS were subjected to H&E staining (top) assessing structural changes and Periodic acid-Schiff staining (PAS; bottom) for mucin production by goblet cells. Boxed area in black borders is zoomed in on the top right corner to show mucin staining (n = 3 for WT and 6 for KO). B: same as in A except formalin-fixed distal colon tissues from WT and Olfr-78 KO mice not treated with DSS were used. C: histological scores from H&E- and PAS-stained sections are presented. ****P < 0.0001.

Fig. 5.

TNF-α treatment reduced olfactory receptor family 51, subfamily E, member 2 (OR51E2) RNA expression in the human colonic cell line. RNA was extracted from control and TNF-α-treated (100 ng/mL) HT-29 colonic cells and analyzed for the expression of OR51E2 by real-time RT-PCR and normalized with GAPDH (n = 3, *P < 0.05).

Fig. 6.

Top: bar plots representing changes in the microbial abundance at phyla level in cecal contents of wild-type (WT) and olfactory receptor-78 (Olfr-78) knockout (KO) mice. Bottom: box plots showing the differences in the abundance of Firmicutes and Bacteriodetes between WT and Olfr-78 KO mice. Differential testing was conducted by two-sample t test. P < 0.05 was considered significant.

Fig. 7.

Validation of few up- and downregulated genes identified by RNA seq analysis of distal colon tissue of wild-type (WT) and olfactory receptor-78 (Olfr-78) knockout (KO) mice. Total RNA was extracted from mucosal scrapings of distal colons from WT and Olfr-78 KO mice and real-time RT-PCR was performed using the gene-specific primers indicated on y-axis and normalized with GAPDH mRNA (n = 3, *P < 0.05 and **P < 0.01).