Elevated granulocytic myeloid-derived suppressor cells are closely related with elevation of Th17 cells in mice with experimental asthma

Abstract

Asthma is a complex and heterogeneous inflammatory response characterized by various immune cells, including myeloid-derived suppressor cells (MDSCs) and CD4⁺ T-cell subsets. However, few studies on MDSC subsets and the association between MDSCs and CD4⁺ T-cell subsets in asthma are reported. In the present study, we detected CD4⁺ T cells and MDSC subsets and evaluated the relationship of these cells in mice with ovalbumin-induced asthma. We found that asthmatic mice showed severe airway inflammatory response and inflammatory cell infiltration in the lungs and bronchoalveolar lavage fluid. We also noted increased numbers of Th2, Th17, and MDSCs; decreased proportion of Th1 and Treg cells in the splenocytes and lungs; and increased expression of pro-inflammatory cytokines in splenocytes and lungs. Granulocytic MDSCs (G-MDSCs) and Th17 cells were closely related. Gemcitabine treatment reduced the G-MDSC level and the iNOS expression, alleviated the inflammatory response, and decreased the proportion and number of Th2 and Th17 cells in asthmatic mice. Besides the increase in Th2 and Th17 cells, the findings indicate that G-MDSC elevation plays a crucial role in asthmatic mice.

Keywords: Th17 cells; experimental asthma; granulocytic myeloid-derived suppressor.

Figure 1

OVA-induced inflammatory response in mice. (A) Experimental scheme. Asthma group was induced by injection with 100 µg of OVA and 2 mg of 10% aluminum hydroxide as adjuvant on days 1, 8, and 15 and challenged by 2% OVA daily from day 22 to day 28. (B) Histological analysis of lung tissue by H&E staining (magnification 400×). (C) Total cells in BALF. (D) Number of eosinophils was calculated in BALF. (E) Number of neutrophils was calculated in BALF. (F) OVA-specific IgE level in sera of mice. The values are mean±SEM (n=12) from two independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 2

Th1/Th2/Th17/Treg cell subset distribution in lungs of mice. The single-cell suspensions of lungs in mice were detected for Th1/Th2/Th17/Treg cell subsets by flow cytometry. (A) CD4⁺IFN-γ⁺ Th1 cells, (B) Th1 cell percentage, (C) Absolute number of Th1 cells, (D) CD4⁺IL-4⁺ Th2 cells, (E) Th2 cell percentage, (F) Absolute number of Th2 cells, (G) CD4⁺IL-17⁺ Th17 cells, (H) Th17 cell percentage, (I) Absolute number of Th17 cells, (J) CD4⁺CD25⁺Foxp3⁺ Treg cells, (K) Treg cell percentage, and (L) absolute number of Treg cells each group are shown. The values are mean±SEM of 12 mice from two independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 3

MDSC subset distribution in splenocytes and lungs of mice. The splenocytes and the single-cell suspensions of lungs in mice were tested for CD11b⁺Ly6G^-Ly6C^high M-MDSC and CD11b⁺Ly6G⁺Ly6C^low G-MDSC subsets by flow cytometry. (A) Representative flow cytometry results are shown in splenocytes of mice. (B) G-MDSC percentage, (C) Absolute number of G-MDSCs, (D) M-MDSC percentage and (E) Absolute number of M-MDSCs in splenocytes of each group are shown. (F) Representative flow cytometry results are shown in lungs of mice. (G) G-MDSC percentage, (H) Absolute number of G-MDSCs, (I) M-MDSC percentage and (J) Absolute number of M-MDSCs in lungs of each group are shown. The values are mean±SEM of 12 mice from two independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 4

Cytokine expression in the spleen and lung tissue of mice. (A) The mRNA expression levels of IFN-γ, IL-4, IL-17A, IL-6, IL-10, and TGF-β in the spleen were evaluated by RT-qPCR. (B) The mRNA expression levels of IFN-γ, IL-4, IL-17A, IL-6, IL-10, and TGF-β in lung tissue were measured through RT-qPCR. The values are mean±SEM of 12 mice from two independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 5

Ratios and relationships among Th1, Th2, Th17, Treg, G-MDSC, and M-MDSC in mice. (A) Ratios of Th2/Treg, (B) Th2/G-MDSC, (C) Th2/M-MDSC, (D) Th17/G-MDSC, and (E) Th17/M-MDSC in each group are shown. The values are mean±SEM of 12 mice from two independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001. (F) Relationship of G-MDSCs with Th2 cells, (G) G-MDSCs with Th17 cells, and (H) G-MDSCs with Treg cells in asthmatic mice. (I) Relationship of Th1 cells with Th2 cells, (J) Th2 cells with Treg cells, and (K) Th17 cells with Treg cells in asthmatic mice (n=8).

Figure 6

Reduction number of MDSCs alleviates inflammation in asthmatic mice. Asthma was induced in mice that were then treated with gemcitabine as described in the Materials and Methods section. The single-cell suspensions of the lungs in mice were tested in terms of CD11b⁺Ly6G^-Ly6C^high M-MDSC and CD11b⁺Ly6G⁺Ly6C^low G-MDSC subsets via flow cytometry. Arg-1 and iNOS expression levels were gated from G-MDSCs and M-MDSCs, respectively. (A) Representative flow cytometry results are shown. (B) The percentage and (C) absolute number of G-MDSCs and the percentage and (E) absolute number of M-MDSCs in each group are shown. (F) Arg-1 and iNOS expression levels in G-MDSCs and M-MDSCs are shown. (G) MFI of Arg-1 in G-MDSCs, (H) MFI of iNOS in G-MDSCs, (I) MFI of iNOS in M-MDSCs, and (J) MFI of iNOS in M-MDSCs in each group are shown. *P < 0.05, **P < 0.01, ***P < 0.001. (K) Histological analysis of the lung section from mice via H&E staining (20× magnification). Images are representatives of two independent experiments (n = 6 mice per group).

Figure 7

Depletion of MDSCs reduces the number of Th2 and Th17 cells in asthmatic mice. Asthma was induced in mice that were then treated with gemcitabine as described in the Materials and Methods section. The single-cell suspensions of the lungs in mice were tested for Th1/Th2/Th17/Treg cell subsets by flow cytometry. (A) CD4⁺IFN-γ⁺ Th1 cells, (B) Th1 cell percentage, (C) Absolute number of Th1 cells, (D) CD4⁺IL-4⁺ Th2 cells, (E) Th2 cell percentage, (F) Absolute number of Th2 cells, (I) CD4⁺IL-17⁺ Th17 cells, (J) Th17 cell percentage, (K) Absolute number of Th17 cells, (L) CD4⁺CD25⁺Foxp3⁺ Treg cells, (M) Treg cell percentage, and (N) absolute number of Treg cells each group are shown. The values are mean±SEM of 12 mice from two independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.