Graphene quantum dots as anti-inflammatory therapy for colitis

Abstract

While graphene and its derivatives have been suggested as a potential nanomedicine in several biomimetic models, their specific roles in immunological disorders still remain elusive. Graphene quantum dots (GQDs) may be suitable for treating intestinal bowel diseases (IBDs) because of their low toxicity in vivo and ease of clearance. Here, GQDs are intraperitoneally injected to dextran sulfate sodium (DSS)-induced chronic and acute colitis model, and its efficacy has been confirmed. In particular, GQDs effectively prevent tissue degeneration and ameliorate intestinal inflammation by inhibiting T_H1/T_H17 polarization. Moreover, GQDs switch the polarization of macrophages from classically activated M1 to M2 and enhance intestinal infiltration of regulatory T cells (T_regs). Therefore, GQDs effectively attenuate excessive inflammation by regulating immune cells, indicating that they can be used as promising alternative therapeutic agents for the treatment of autoimmune disorders, including IBDs.

Fig. 1. Intraperitoneal injection of GQDs effectively alleviates DSS-induced chronic colitis in mice.

(A) Experimental scheme for DSS-induced chronic colitis and GQD administration. DSS water (3.5%) was repetitively administered to mice to induce colitis. GQDs were injected intraperitoneally (300 μg per head) 10 days after the administration of DSS. (B to F) Mice received intraperitoneal injection of GQDs after chronic DSS colitis induction. On day 27, mice were sacrificed for further investigation. (B) The percentage of body weight change and (C) the DAI for colitis severity were monitored for clinical assessment. (D) After 27 days from colitis induction, the lengths of the colons obtained from each group were measured. Photo credit: Byung-Chul Lee (Adult Stem Cell Research Center and Research Institute for Veterinary Science, College of Veterinary Medicine, Seoul National University). (E) Left: Representative images of colon sections stained with hematoxylin and eosin (H&E) and picrosirius red (PSR) staining for the assessment of histology and fibrosis. Scale bar, 200 μm. Right: Histopathologic evaluations were conducted to examine lymphocyte infiltration and intestinal damage. Quantitative analysis of the fibrotic area (stained in red). (F) Serum was collected from the colitis mice, and the secreted levels of the indicated cytokines were assessed using CBA analysis. N = 4 to 5 mice per group. *P < 0.05, **P < 0.01, and ***P < 0.001. Results are shown as means ± SD.

Fig. 2. Intraperitoneal injection of GQDs suppresses DSS-induced acute colitis in mice.

(A) Experimental scheme for acute colitis induction using DSS and administration of GQDs. (B to H) Mice received intraperitoneal injection of GQDs after acute DSS colitis induction. On day 14, mice were sacrificed for further investigation. (B) The survival rate and (C) percentage of body weight change were monitored for clinical assessment of colitis severity (N = 14 to 16 mice per group). (D) The DAI on day 10 were monitored. (E) Mice were sacrificed 14 days after the induction of colitis with DSS, and colon lengths were measured to determine intestinal damage. Photo credit: Byung-Chul Lee (Adult Stem Cell Research Center and Research Institute for Veterinary Science, College of Veterinary Medicine, Seoul National University). (F) The MPO level in the colon tissues was measured. (G) Left: Representative images of colon sections stained with H&E and Masson’s trichrome (MT) staining for the assessment of fibrosis. Scale bar, 200 μm. Right: Histopathologic evaluations were conducted to assess lymphocyte infiltration and intestinal damage. Quantitative analysis of fibrotic area (stained in blue). (H) Serum was collected from the colitis mice, and the secreted levels of the indicated cytokines were assessed using CBA analysis (N = 5 mice per group). *P < 0.05, **P < 0.01, and ***P < 0.001. Results are shown as means ± SD.

Fig. 3. GQDs are excreted from mice without generating toxicity.

Without DSS induction, biotin-labeled GQDs were injected into normal mice by the same method and dosage, and monitored for 16 weeks (N = 5 mice per group). (A) Body weights and (B) water and food consumption were measured at each time point. (C) Mice were sacrificed at the indicated time points, and organ weights were assessed. (D) The FITC-labeled anti-biotin antibody was used to detect the presence of GQDs in the abdominal mesenteric fat. (E) Excretion of GQDs was investigated in urine collected from the mice. *P < 0.05 and ***P < 0.001. Results are shown as means ± SD.

Fig. 4. GQDs suppress T_H1-type immune responses.

(A and B) Colons and serum samples were collected from chronic colitis–induced mice after administration of GQDs (N = 4 to 5 mice per group). (A) Proportions of CD4 and IFN-γ–expressing T_H1 cells in the colon were analyzed by flow cytometric analysis. (B) IL-12 secretion levels in the serum were measured by CBA analysis. (C) Primary CD4⁺ T cells were isolated from human cord blood and cultured in the presence of anti-CD3/28 microbeads and GQDs for 2 days, and proliferation was assessed with a BrdU assay. OD, optical density. (D to G) Human primary CD4⁺ T cells were induced to differentiate into T_H1 cells using anti-CD3/28 beads in combination with recombinant IL-2, IL-12, and IFN-γ in the presence of GQDs for 5 days. (D and E) The proportion of CD4⁺IFN-γ⁺ cells was determined by flow cytometric analysis. (D) Representative dot plot images. (E) Histogram of collective data. (F) mRNA expression levels in each group of induced T_H1 cells were investigated for the indicated T_H1-specific markers. (G) The indicated T_H1-specific cytokines in the supernatant of T_H1 cells were analyzed by CBA analysis (n = 4 to 5). *P < 0.05, **P < 0.01, and ***P < 0.001. Results are shown as means ± SD.

Fig. 5. The effects of GQDs on cell fate decisions of T_regs and T_H17 cells.

(A, D, and F to H) The GQD-treated mice with chronic colitis were sacrificed, and colons and serum samples were collected for further ex vivo examination (N = 4 to 5 mice per group). (A) Colonic infiltration of CD4⁺CD25⁺FoxP3⁺ T_regs was assessed by flow cytometric analysis. (B and C) The GQD-treated mice with acute colitis were sacrificed, and colons were collected for further ex vivo examination (N = 8 to 12 mice per group). DAPI, 4′,6-diamidino-2-phenylindole. (B) T_regs in the colon were observed by immunostaining of FoxP3 (green). FoxP3⁺ cells are indicated as arrows. Scale bar, 50 μm. (C) TGF-β1 expression in the colons was detected by ELISA. (D) IL-10 level was determined by CBA analysis. (E) CD4⁺ T cells were isolated from human cord blood, polarized into T_regs with specific cytokines, and the proportions of CD4⁺CD25⁺FoxP3⁺ cells were determined by flow cytometric analysis (n = 5). (F) Proportions of T_H17 cells in the spleens were analyzed. The indicated T_H17-specific cytokines, (G) IL-17A, and (H) IL-23 in the serum were analyzed. *P < 0.05, **P < 0.01, and ***P < 0.001. Results are shown as means ± SD.

Fig. 6. GQDs promote M2 polarization of macrophages.

(A and B) Primary CD14⁺ macrophage-like cells were polarized into M1-type cells. (A) CD14⁺ macrophage-like cells were isolated from human cord blood and polarized into M0-, M1-, and M2-type cells with specific inducer cytokines in the presence of GQDs. The cells were immunostained for the cell surface markers CD14 and CD206. Scale bar, 50 μm. (B) M1-polarized macrophages were analyzed by the type-specific cell surface CD markers CD86, CD206, and CD163 using flow cytometry. (C) The indicated cytokine concentrations were measured in supernatants of M1 macrophages. (D) M1-induced cells in the presence of GQDs were cocultured with naïve CD4⁺ T cells supplemented with IL-2 and TGF-β1, and the proportions of T_regs were investigated by flow cytometry. (E) In vitro M2b polarization was analyzed by flow cytometric analysis (n = 3). (F) The presence of M2b macrophages in the peritoneum of chronic colitis–induced mice was detected by flow cytometric analysis (N = 4 to 5 mice per group). (G) Schematic diagram for the present study. **P < 0.01 and ***P < 0.001. Results are shown as means ± SD.