Combinations of anti-GITR antibody and CD28 superagonist ameliorated dextran sodium sulfate-induced mouse colitis

Abstract

Ulcerative colitis (UC) is one of the two main forms of inflammatory bowel disease (IBD) and is an idiopathic, chronic inflammatory disease of the colonic mucosa with an unclear etiology. Interleukin (IL)-10 has been reported to play a crucial role in the maintenance of immune homeostasis in the intestinal environment. Type 1 regulatory T (Tr1) cells are a subset of CD4+Foxp3- T cells able to secrete high amounts of IL-10 with potent immunosuppressive properties. In this study, we found that the combination of anti-GITR antibody (G3c) and CD28 superagonist (D665) treatment stimulated the generation of a large amount of Tr1 cells. Furthermore, G3c/D665 treatment not only significantly relieved severe mucosal damage but also reduced the incidence of colonic shortening, weight loss, and hematochezia. Dextran sodium sulfate (DSS) upregulated the mRNA levels of IL-6, IL-1β, IL-17, IL-12, tumor necrosis factor-alpha, C-C chemokine receptor type 5, and Bax in splenic lymphocytes (SPLs) and colon tissues, while G3c/D665 treatment conversely inhibited the increase in mRNA levels of these genes. In addition, G3c/D665 treatment altered the proportion of CD4+ and CD8+ T cells and increased CD4+CD25+Foxp3+ regulatory T cells in SPLs, mesenteric lymph nodes (MLNs), and lamina propria lymphocytes (LPLs). Thus, the combination of G3c and D665 treatment showed efficacy against DSS-induced UC in mice by inducing a large amount of Tr1 cell generation via the musculoaponeurotic fibrosarcoma pathways in vivo and relieving inflammatory responses both systematically and locally.

Keywords: CD28 superagonist; anti-GITR antibody; dextran sulfate sodium; inflammatory bowel disease; type 1 regulatory T cells.

Graphical Abstract

Figure 1:

G3c/D665 treatment attenuated DSS-induced damage of colon. (A) The colon gross appearance and length analysis of mice are shown. The DAI of mice and the weight change (%) of mice are also presented. (B) Hematoxylin and eosin (HE) staining of colon specimens in the three groups. Substantial monocyte infiltration, mucosa erosion, goblet cell arrangement disorder, and reduction were seen in the colon specimens of the 3.5% DSS-control group. A Hematoxylin and eosin (HE) analysis of the colon specimens is shown (scale bars = 100 and 200 µm). Each bar represents the mean ± SD. *P < 0.05, ****P < 0.0001.

Figure 2:

G3c/D665 treatment reduced the mRNA expression of inflammatory and apoptosis cytokine-related genes. (A) Homogenates of colon tissues were analyzed by qRT-PCR as described in the materials and methods. The mRNA expression of inflammatory cytokines-related genes, particularly IL-1β, IL-6, IL-12, IL-17, and CCR-5, was significantly lower in the G3c/D665-treated group than in the control group. The mRNA expression of apoptosis-related gene, such as Bax, was reduced in the G3c/D665-treated group. (B) SPLs were analyzed by qRT-PCR as well. Compared with the control group, the mRNA expression of inflammatory and apoptosis cytokine-related genes, such as IL-1β, IL-12, and Bcl-2, was reduced in the G3c/D665-treated group. Values are expressed as arbitrary units (mean ± SD). *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 3:

G3c/D665 treatment increased the proportion of CD4⁺ T cells in SPLs, MLNs, and LPLs. (A) A representative FCM analysis assessing the expression of CD8⁺ and CD4⁺ T cells gated on CD3⁺ T cells in SPLs, MLNs, and LPLs of three groups. (B) The proportion of CD4⁺ T cells was significantly increased in the G3c/D665-treated group compared to the control group in SPLs, MLNs, and LPLs. The proportion of CD8⁺ T cells was significantly reduced in the G3c/D665-treated group compared to the 3.5% DSS group in SPLs and MLNs. Data are expressed as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 4:

G3c/D665 treatment increased the proportion of CD4⁺CD25⁺Foxp3⁺ Tregs in SPLs, MLNs, and LPLs. (A) A representative FCM analysis assessing the expression of CD4⁺CD25⁺Foxp3⁺ Tregs gated on CD4⁺ T cells in the SPLs, MLNs, and LPLs of the three groups. (B) The proportion of Tregs was significantly increased in the G3c/D665-treated group compared to the control group among SPLs, MLNs, and LPLs. (C–D). A representative FCM analysis assessing the MFI of CTLA-4 expression in the SPLs, MLNs, and LPLs of the three groups gated from CD4⁺CD25⁺Foxp3⁺ Tregs. Data are expressed as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 5:

G3c/D665 treatment increased the proportion of IL-10⁺IFN-g⁺Tr1 cells and IL-10⁺Foxp3^- Tr1 cells in SPLs, MLNs, and LPLs. (A) A representative FCM analysis assessing the proportion of IL-10⁺IFN-g⁺ Tr1 and IL-10⁺Foxp3^- Tr1 cells gated on CD4⁺ T cells among the SPLs, MLNs, and LPLs of the three groups. (B) The proportion of IL-10⁺IFN-g⁺ Tr1 cells and IL-10⁺Foxp3^- Tr1 cells was significantly increased in the G3c/D665-treated group compared to the control group among SPLs, MLNs, and LPLs. Data are expressed as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 6:

G3c/D665 treatment altered the mRNA expression and protein levels of Tr1 cell-related genes. MLNs (A), SPLs (B) and colon tissues (C) were analyzed by qRT-PCR as described in the materials and methods. The mRNA expression of Tr1 cell-related genes, particularly Prdm1, IL-10, Eomes, and Ahr is shown. (D). A Western blot analysis of the Maf of the SPLs in the three group is shown. Data are expressed as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 7:

Immune mechanism of G3c/D665 in the treatment of UC. In UC, the intestinal barrier is disrupted, the mucosa is severely damaged, and the balance of intestinal microflora is dysregulated. In the colonic lamina propria, Th1 cells release many pro-inflammatory cytokines, such as IL-1β, TNF-α, IL-6, IL-12, and CCR-5, while Th17 cells release IL-17. The release of apoptotic factor, such as Bax, is also increased in UC. G3c and D665 treatment expands Tregs and Tr1 cells, thereby increasing the release of anti-inflammatory cytokines and decreasing the release of pro-inflammatory cytokines and apoptotic factors. Tregs release IL-10 and CTLA-4, while Tr1 cells release IL-10, Prdm1, Eomes, and Maf. After the administration of G3c and D665, the intestinal barrier is restored, mucosal damage is repaired, and colitis is improved.