Gr-1⁺CD11b⁺ immature myeloid cells (IMC) promote resistance of pro-inflammatory T cells to suppression by regulatory T cells in atherosclerotic Apo E- deficient mice

Abstract

Accumulating evidence indicates that both defects in Treg numbers and/or function as well as resistance of effector T cells to suppression may contribute to the development of human chronic inflammatory diseases. However, which mechanism involved in the progression of atherosclerosis remains unclear. In this study, we evaluated the production and function of CD4⁺ inflammatory and regulatory T cells in atherosclerosis-prone mice. We found that the hyperactivity and unresponsiveness to Treg-mediated suppression of inflammatory CD4⁺ T cells occurred in the progression of atherosclerosis, though Treg cells were present in very large numbers and fully functional. We further found that Gr-1⁺CD11b⁺ immature myeloid cells were significantly accumulated in atherosclerotic Apo E⁻/⁻ mice, and they promoted resistance of inflammatory CD4⁺ T cells to Treg-mediated suppression in vitro and in vivo. we further confirmed that Gr-1⁺CD11b⁺ immature myeloid cells produced high level of interleukin 6 which was at least partially responsible for inducing unresponsiveness of inflammatory CD4⁺ T cells to suppression via activation of Jak/Stat signaling pathway. Taken together, these findings might provide new insights to explore potential targets for immune therapeutic intervention in atherosclerosis.

Figure 1. The frequencies of both Th1 and Th17 cells in the spleen of Apo E^−/− mice increase in parallel to the rise in the serum level of total cholesterol and IL-6.

(a), the serum level of Total cholesterol levels in Apo E^−/− mice were measured compared with age-matched C57BL/6 mice. Data are the mean ± SEM (n = 4) of one representative experiment. **p<0.01, ***p<0.001. (b), the serum level of IL-6 in Apo E^−/− mice and age-matched C57BL/6 mice was measured by ELISA. Shown is one representative experiment of three performed. *p<0.05, **p<0.01. Apo E^−/− mice were fed with standard chow diet and sacrificed at age 6, 12, 24, and 48 weeks. Splenocytes were stained with FITC-CD4, PE-IL-17A and APC-IFN-γ antibody, (c), Representative plots are shown. The frequencies of Th1and Th17 cells are shown in (d) and (e), respectively. Data are the mean ± SEM (n = 4) of one representative experiment out of three performed. **p<0.01, ***p<0.001.

Figure 2. The accumulation of Treg and IMC in the spleen of Apo E^−/− mice.

(a), Splenocytes were stained with FITC-CD4 and PE-FoxP3 antibody, the frequencies of Treg cells at time points mentioned above were measured by flow cytometry. Representative plots are shown in (b). Data are the mean ± SEM (n = 4) of one representative experiment out of three performed. (c), Splenocytes were stained with FITC-Gr-1 and PE-CD11b antibody, the frequencies of IMC in the spleen at indicated time points were measured by flow cytometry. Representative plots are shown in (d). Data are the mean ± SEM (n = 4) of one representative experiment out of three performed. n.s = not significant, *p<0.05, **p<0.01.

Figure 3. Resistance of pro-inflammatory T cells to suppression, instead of impaired Treg cells, contributes to the ongoing inflammatory response in atherosclerotic Apo E^−/− mice.

(a), 6×10⁴ CD4⁺CD25⁻ T cells from C57BL/6 mice were stimulated with 3 µg/ml CD3 and 1 µg/ml CD28, in the presence or not of indicated numbers of Treg from 20-week old Apo E^−/− mice or age-matched C57BL/6 mice for 3 days. 1 µCi of [3H] thymidine was added in each well 18 hrs before harvest, T cell proliferation was determined by [3H] thymidine incorporation. Data are the mean ± SEM (n = 4) of one representative experiment out of three performed. CD4⁺CD25⁻ T cells isolated from 20-week old Apo E^−/− mice or age-matched C57BL/6 mice were cultured alone or with Treg cells isolated from 20-week old Apo E^−/− mice at 3∶1 ratio, T cell proliferation was examined as shown in (b). The amounts of IFN-γ (c) and IL-17A (d) in supernatant were measured by ELISA (n = 4). Data are representative of three independent experiments. n.s = not significant; *p<0.05, **p<0.01, ***p<0.001.

Figure 4. Accumulated Gr-1⁺CD11b⁺ cells cause unresponsiveness of pro-inflammatory T cells to suppression by Treg cells in Apo E^−/− mice.

(a), CD4⁺CD25⁻ T cells from C57BL/6 mice were cultured with or without Gr-1⁺CD11b⁺ cells and Treg cells, T cell proliferation was determined by [3H] thymidine incorporation. Similar results were obtained in three independent experiments. (b), 6×10⁴ CD4⁺CD25⁻ T cells from C57BL/6 mice were cultured in the presence of indicated numbers of Gr-1⁺CD11b⁺ cells from 20-week old Apo E^−/− mice for 3 days. 1 µCi of [3H] thymidine was added in each well 18 hrs before harvest, T cell proliferation was determined by [3H] thymidine incorporation. Data are the mean ± SEM (n = 4) of one representative experiment out of three performed. (c), atherosclerotic Apo E^−/− mice were pre-treated i.p. with 100 µg RB6-8C5 mAb or PBS twice a week before adoptive transfer of 1×107 CFSE-labeled C57BL/6 CD4⁺CD25⁻ T cells. Three days after transfer, mice were sacrificed and CFSE dilution was analysis by flow cytometry. Data are the mean ± SEM (n = 5) of one representative experiment. Similar results were obtained in at least three independent experiments. n.s = not significant; *p<0.05, **p<0.01.

Figure 5. Gr-1⁺CD11b⁺ cell-induced unresponsiveness of pro-inflammatory T cells to suppression is IL-6 dependent.

(a), Gr-1⁺CD11b⁺ cells were isolated from the spleen of 20-week old Apo E^−/− and C57BL/6 mice and EL-4 tumor-bearing mice. The mRNA expression of Arg 1, iNOS 2, TGF-β1 and IL-6 was measured by qRT-PCR. (b), CD4⁺CD25⁻ T cells from C57BL/6 mice were cultured with or without Gr-1⁺CD11b⁺ cells and Treg cells as well as 20 µg/ml IL-6 antibody, T cell proliferation was determined by [3H] thymidine incorporation. Data are the mean ± SEM (n = 4) of one representative experiment. Atherosclerotic Apo E^−/− mice were pre-treated i.p. with 100 µg RB6-8C5 mAb or Jak inhibitor tofacitinib (CP-690,550) 30 mg/kg twice a week, (c), Phosphorylation of stat1 and stat3 as well as Erk was detected by immunoblot. Shown is one representative experiment out of three. the serum levels of IL-6 (d), INF-γ (e), IL-17A (f) and TGF-β1 (g) were measured by ELISA. Data are the mean ± SEM (n = 4) of one representative experiment. Similar results were obtained in at least three independent experiments. n.s = not significant; *p<0.05, **p<0.01.