Gpr174 Knockout Alleviates DSS-Induced Colitis via Regulating the Immune Function of Dendritic Cells

Abstract

Background: Dysfunction of the immune system would disturb the intestinal homeostasis and lead to inflammatory bowel disease (IBD). Dendritic cells (DCs) help maintain intestinal homeostasis and immediately respond to pathogens or injuries once the mucosa barriers are destroyed during IBD. G protein-coupled receptors(GPR)174 is an essential regulator of immunity that is widely expressed in most immune cells, including DCs. However, the role of GPR174 in regulating the immune function of DC in colitis has not been investigated.

Methods: Dextran sodium sulfate (DSS) was administered to establish the mice colitis model. Data of weight, length of colon, disease activity index (DAI), and macroscopic scores were collected. The flow cytometry was used to detect the infiltrations of T cells and DCs, the mean fluorescence intensity (MFI) of CD80, CD86, CD40, and major histocompatibility complex-II (MHC-II). And T cells proliferataion was measured by carboxyfluorescein diacetate succinimidyl ester (CFSE). The expression of cytokines (tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-10 (IL-10), interferon-γ (IFN-γ), interleukin -4 (IL-4)) and GPR174 mRNA were measured by Elisa, quantitative polymerase chain reaction (qPCR), and immunofluorescence. RNA of bone-marrow-derived dendritic cells (BMDCs) was extracted for sequencing. Adoptive transfer of BMDCs was administrated intravenously.

Results: Gpr174^-/- mice exposed to 3% DSS showed significant alleviation characterized by reduced loss of weight, more minor colon damage, and better DAI and macroscopic scores. The expression of pro-inflammatory cytokines (TNF-α, IL-6) decreased, while anti-inflammatory cytokine (IL-10) increased compared with WT mice. In vitro, Gpr174^-/- BMDCs showed less maturity, with a declined expression of MHC-II, CD80, CD86 and reduced TNF-α, higher IL-10 after LPS stimulation. Gpr174^-/- BMDCs were less capable of activating OT-II naïve CD4⁺ T cells than WT BMDCs and induced more Th0 cells to differentiate into Treg while less into Th1. Furthermore, the transcriptome sequencing analysis exhibited that Gpr174 participated in TNF-α (NF-κB) signaling, leukocyte transendothelial migration, and Th1/Th2 cell differentiation pathways. Adoptive transfer of Gpr174^-/- BMDCs to WT mice ameliorated DSS-induced colitis.

Conclusion: Our study indicated that GPR174 was involved in the pathogenesis of IBD by regulating the maturation of the dendritic cells to maintain immune homeostasis. TNF-α (NF-κB) signaling pathway, leukocyte transendothelial migration, and Th1/Th2 cell differentiation pathways may be the target pathway.

Keywords: GPR174; T cell activation; dendritic cells; inflammatory bowel disease; intestinal barrier function.

Figure 1

Gpr174^-/- mice were resistant to DSS-induced colitis. (A) Body weight loss in each group, (B) DAI scores in each group, (C, D) Colon length of each group, (E) Macroscopic score of each group, (F) Morphology and HE of the intestine of each group. Data were exhibited as mean ± SEM of 5 mice per group. (Black arrow: epithelial barrier disruption. *P < 0.05, **P < 0.01).

Figure 2

Gpr174 knockout reduced intestinal inflammation and protected the intestinal barrier. (A) Intestinal MPO expression in each group, (B) Intestinal cytokine expression of TNF-α, IL-6, IL-10 in each group, (C, D) Intestinal tight junctions of Zo-1 and Occludin mRNA expression in each group, (E) Intestinal permeability in each group. Data were exhibited as mean ± SEM of 5 mice per group. (*P < 0.05, **P < 0.01, ***P < 0.001).

Figure 3

Gpr174 knockout increased CD11c⁺ dendritic cells infiltration while suppressed maturation in the lamina propria. (A, B) Infiltration of CD11c⁺ dendritic cells in the lamina propria in each group, (C) MHC-II expression of CD11c⁺ dendritic cells in each group. Data were exhibited as mean ± SEM of 5 mice per group. (*P < 0.05).

Figure 4

Gpr174 knockout inhibited maturation of BMDCs. (A) CD11c⁺ expression of BMDCs in each group, (B) Co-stimulatory molecular MHC-II, CD80, CD86, CD40 BMDCs. Data were exhibited as mean ± SEM of 5 mice per group.

Figure 5

Gpr174 knockout inhibited T cell proliferation and influenced T cell differentiation. (A) Degree of naive T cells proliferation, which was labeled as CFSE after stimulation in each group, (B) IFN-γ expression of naive CD4⁺ T cells after stimulation in each group, (C) IL-4 expression of naive CD4⁺ T cells after stimulated in each group, (D) IL-10 expression of naive CD4⁺ T cells after stimulated in each group. Data were exhibited as mean ± SEM of 5 mice per group. (*P < 0.05, **P < 0.01).

Figure 6

Adoptive transfer of Gpr174^-/- BMDCs alleviated DSS-induced colitis. (A) Body weight loss in each group after adoptive transfer of Gpr174^-/- DC, (B) DAI scores in each group, (C) Flow chart of the procedure of adoptive transfer of BMDCs and establishing DSS-induced colitis animal models, (D) Macroscopic score of each group, (E, F) Colon length of each group, (G) Morphology and HE of the intestine of each group. Data were exhibited as mean ± SEM of 5 mice per group. (*P < 0.05, **P < 0.01).

Figure 7

Adoptive transfer of Gpr174^-/- BMDCs alleviated inflammatory response of colitis. (A) Colonic MPO expression in each group, (B) Colonic cytokine expression of TNF-α in each group, (C) Intestinal cytokine expression of IL-6 in each group, (D) Intestinal cytokine expression of IL-10 in each group, (E) CD3⁺ T cells infiltration in the lamina propria in each group (*P < 0.05, ***P < 0.001).

Figure 8

Gpr174 knockout influences BMDCs transcriptome after LPS stimulation. (A) Venn diagram of differential expression genes (DEGs) of four groups of BMDCs before and after LPS application, (B) Heatmap of DEGs of BMDCs in each group after LPS application, (C) Protein-protein interactions (PPI) networks analysis of DEGs of Gpr174^-/- BMDCs and Gpr174^+/+ BMDCs after LPS application, (D) KEGG analysis of DEGs of Gpr174^-/- BMDCs and Gpr174^+/+ BMDCs after LPS application (the first twenty 20 pathways with ease < 0.05, count ≥ 10, and FDR < 0.01).

Figure 9

Expression of GPR174 between healthy and pathogenic tissues. (A) The mRNA expression of GPR174 between healthy and pathogenic tissues from the ulcerative patients(n=10), (B) The protein expression of GPR174 receptor between healthy and pathogenic tissues from the ulcerative patients (Blue: DAPI; Green: GPR174). (*P < 0.05).