Atorvastatin promotes the expansion of myeloid-derived suppressor cells and attenuates murine colitis

Abstract

Statins, widely prescribed as cholesterol-lowering drugs, have recently been extensively studied for their pleiotropic effects on immune systems, especially their beneficial effects on autoimmune and inflammatory disorders. However, the mechanism of statin-induced immunosuppression is far from understood. Here, we found that atorvastatin promoted the expansion of myeloid-derived suppressor cells (MDSCs) both in vitro and in vivo. Atorvastatin-derived MDSCs suppressed T-cell responses by nitric oxide production. Addition of mevalonate, a downstream metabolite of 3-hydroxy-3-methylglutaryl coenzyme A reductase, almost completely abrogated the effect of atorvastatin on MDSCs, indicating that the mevalonate pathway was involved. Along with the amelioration of dextran sodium sulphate (DSS) -induced murine acute and chronic colitis, we observed a higher MDSC level both in spleen and intestine tissue compared with that from DSS control mice. More importantly, transfer of atorvastatin-derived MDSCs attenuated DSS acute colitis and T-cell transfer of chronic colitis. Hence, our data suggest that the expansion of MDSCs induced by statins may exert a beneficial effect on autoimmune diseases. In summary, our study provides a novel potential mechanism for statins-based treatment in inflammatory bowel disease and perhaps other autoimmune diseases.

Keywords: atorvastatin; immunosuppression; murine colitis; myeloid-derived suppressor cells; nitric oxide.

Figure 1

Atorvastatin enhanced the expansion of myeloid‐derived suppressor cells (MDSCs) in vitro. (a) Mouse bone marrow (BM) cells were cultured in the presence of granulocyte–macrophage colony‐stimulating factor (GM‐CSF) (20 ng/ml) and 5 μm atorvastatin. DMSO was used as control. The frequency of MDSCs (CD11b⁺ Gr‐1⁺), MDSCs subsets, and dendritic cells (DCs) (CD11c⁺ MHC‐II⁺) were measured by flow cytometry analysis. Representative data from a single experiment (left), as well as mean + SEM of three independent experiments (right) are shown. (b) The suppressive function of atorvastatin‐derived and control granulocytic (G‐) MDSCs (CD11b⁺ Gr‐1^high) was determined. C57BL/6 mouse BM cells were cultured in the presence of GM‐CSF and interleukin‐6 (IL‐6) (20 ng/mL) for 5 days with atorvastatin or DMSO control. G‐MDSCs (CD11b⁺ Gr‐1^high) were purified by flow cytometric sorting. Allogeneic CD3⁺ T cells containing CD4⁺ and CD8⁺ T cells (from BALB/c mice) were stimulated with anti‐CD3/CD28 antibodies, and co‐cultured with isolated G‐MDSCs at different ratios for 3 days. CD4⁺ and CD8⁺ T‐cell proliferation was determined by CFSE dilution. Unstimulated T cells were used as a negative control. Representative data from a single experiment (left), as well as mean+SEM of three independent experiments (right) are shown. Ator, atorvastatin. *P < 0·05, **P < 0·01.

Figure 2

Atorvastatin promoted granulocytic myeloid‐derived suppressor cell (G‐MDSC) accumulation in vivo. (a) C57BL/6 mice (6–8 weeks) were injected intraperitoneally with atorvastatin at a dose of 50 mg/kg/day in a volume of 50 μl 5% DMSO/castor oil (n = 6). Control mice received the vehicle (5% DMSO/castor oil) via the same route (n = 6). Mice were killed after 12 days and different tissues were obtained. Proportions of MDSC subsets in the spleen, bone marrow (BM), liver and peripheral blood mononuclear cells (PBMCs) were measured by flow cytometry. Representative data from a single mouse (left), as well as the mean + SEM from all mice (right) are shown. (b) The suppressive function of G‐MDSCs (CD11b⁺ Gr‐1^high) from the spleen of mice treated with atorvastatin. Unstimulated T cells were used as a negative control. Representative data from a single experiment (left), as well as mean + SEM of three independent experiments (right) are shown. **P < 0·01.

Figure 3

Atorvastatin‐derived granulocytic myeloid‐derived suppressor cells (G‐MDSCs) inhibited T‐cell responses by nitric oxide production. (a) G‐MDSCs or immature myeloid cells (IMC) were isolated from the spleens of mice treated with atorvastatin or vehicle control. The expression of l‐arginine metabolizing enzymes was measured by quantitative RT‐PCR. (b) Effect of different inhibitors on the function of atorvastatin‐derived G‐MDSCs was evaluated by T‐cell proliferation assay. CD3⁺ T cells containing CD4⁺ and CD8⁺ T cells from BALB/C mice were stimulated with anti‐CD3/CD28 antibodies and co‐cultured with allogeneic MDSCs isolated from the spleen of mice treated with atorvastatin at a 2 : 1 ratio for 3 days with treatments as indicated. (c) The culture supernatant was collected for NO content determination. NW‐hydroxy‐nor‐arginine (nor‐NOHA) (100 μm): arginase inhibitor; l‐arginine (1 mm); l‐NG‐monomethyl‐arginine (l‐NMMA (100 μm): iNOS inhibitor; N‐acetyl‐l‐cysteine (NAC) (1 mm): reactive oxygen species (ROS) inhibitor. (d) ROS production in MDSCs was measured by flow cytometry. CD11b⁺ Gr‐1^high cells were gated and the percentage of CM‐H2DCFDA ⁺ cells is shown. (e) Effect of mevalonate on the MDSCs expansion mediated by atorvastatin. Mouse bone marrow cells were cultured in the presence of granulocyte–macrophage colony‐stimulating factor (GM‐CSF) + interleukin‐6 (IL‐6) with treatments as indicated. After 5 days, the frequency of MDSCs was evaluated by flow cytometry. Meva (100 μm), mevalonolactone. Representative data from a single experiment (left) are shown. For all the experiments, mean + SEM of three independent experiments is shown. *P < 0·05, **P < 0·01.

Figure 4

Atorvastatin attenuated dextran sodium sulphate (DSS) ‐induced murine colitis with myeloid‐derived suppressor cell (MDSC) expansion. (a–f) C57BL/6 mice were injected intraperitoneally with atorvastatin (50 mg/kg/day). At day 3, mice were given water containing 2·5% DSS or drinking water (a). Mice were killed at day 10 for evaluation of colitis severity: weight loss (b), the colon length (c), disease activity index (DAI) (d), colon histology (100 ×) (e) and histology score (f). (g) Proportions of MDSCs (CD11b⁺ Gr‐1⁺) in different tissues of mice were evaluated by flow cytometry. Representative graph (left) and statistical graph (right) are shown (n = 6). (h) MDSC subsets in mouse spleen and colon lamina propria mononuclear cells (LPMCs) were evaluated by flow cytometry. For (b) and (d), the DSS+Ator group was compared with the DSS group. *P < 0·05, **P < 0·01.

Figure 5

Atorvastatin‐derived granulocytic myeloid‐derived suppressor cells (G‐MDSCs) ameliorated dextran sodium sulphate (DSS) ‐induced murine colitis. (a) The experimental design. Enriched G‐MDSCs (2 million) from the spleen of mice treated with atorvastatin were injected into recipient mice via the tail vein. At day 8, mice were killed for examination. (b) Analysis of MDSC subsets in peripheral blood mononuclear cells (PBMCs) and spleens of mice without DSS treatment after G‐MDSC transfer. The murine colitis severity was evaluated as in Fig. 4: weight loss (c), disease activity index (DAI) (d), the colon length (e), representative colon histology (100 ×) (f) and histology score (g). (h) The representative flow cytometry plots of MDSC subsets (with CD11b⁺ cells pre‐gated) in spleen and colon lamina propria mononuclear cells (LPMCs) for all four groups. For (c) and (d), DSS+Ator‐MDSC group was compared with the DSS+Ctrl‐IMC group. Ctrl, control; Ator, atorvastatin. *P < 0·05, **P < 0·01.

Figure 6

Atorvastatin attenuated dextran sodium sulphate (DSS) ‐induced murine chronic colitis. (a) The experimental design. Mice were killed at day 55. Chronic colitis was evaluated by: weight loss (b), disease activity index (DAI) (c), colon length (d) and colon histology (100 ×) and histology score (e). (f) Proportions of myeloid‐derived suppressor cells (MDSCs) (CD11b⁺ Gr‐1⁺) in different tissues of mice were evaluated by flow cytometry. Representative graph (left) and statistical graph (right) are shown (n = 5). (g) The representative flow cytometry plots of MDSCs subsets (with CD11b⁺ cells pre‐gated) in spleen and colon lamina propria mononuclear cells (LPMCs) for DSS and DSS+Ator treatment. For (b) and (c), DSS+Ator group was compared with DSS group. *P < 0·05, **P < 0·01.

Figure 7

Atorvastatin‐derived granulocytic myeloid‐derived suppressor cells (G‐MDSCs) attenuated the T‐cell transfer of chronic colitis. Rag1 ^−/− mice were injected intraperitoneally with 5 × 10⁵ CD4⁺ CD45RB ^high T cells from C57BL/6 mice, 5 × 10⁵ CD4⁺ CD45RB ^high T cells + 5 × 10⁵ G‐MDSCs or 5 × 10⁵ immature myeloid cells (IMCs) with the same phenotype from the spleens of mice after atorvastatin or vehicle treatment. Mice were killed after 8 weeks. (a) Body weight loss of mice with different treatment. (b) Colon length of mice after 8 weeks adoptive transfer. (c) Representative colon histology (100 ×) and histology score. (d) Absolute numbers of CD4⁺ T cells in the spleen, mesenteric lymph nodes (mLNs) and colon lamina propria mononuclear cells (LPMCs) *P < 0·05, **P < 0·01.