Chronic psychological stress induces the accumulation of myeloid-derived suppressor cells in mice

Abstract

Chronic psychological stress has been shown to adversely impact immune system functions and compromise host defenses against various infections. However, the underlying mechanisms remain elusive. Recent studies have demonstrated that myeloid-derived suppressor cells (MDSCs) play an important role in regulating immunity. It is of interest to explore whether or not chronic psychological stress plays immunosuppressive functions partially by inducing MDSCs accumulation. In this work, we report that chronic psychological stress led to the accumulation of CD11b+Gr1+ cells in the bone marrow of BALB/c mice. Repeated β-agonist infusion showed no such effect. However, β-adrenergic blockade, but not glucocorticoids blockade, partially reversed the accumulation of CD11b+Gr1+ cells under the condition of chronic psychological stress, suggesting catecholamines collaborate with other factors to induce the accumulation. Further exploration indicates that cyclooxygenase 2 (COX-2)-prostaglandin E2 (PGE2) loop might act downstream to induce the accumulation. A majority of the accumulated CD11b+Gr1+ cells were Ly6G+Ly6C(low) immature neutrophils, which inhibited cytokine release of macrophages as well as T cell responsiveness. Moreover, the accumulated CD11b+Gr1+ cells under the condition of chronic psychological stress expressed multiple inhibitory molecules. Taken together, our data demonstrate for the first time that chronic psychological stress induces MDSCs accumulation in mice, which can contribute to immunosuppression.

Figure 1. Repeated restraint sessions led to reduced organ weight and cell numbers.

BALB/c mice were subjected to 5-days restraint stress or left untreated, n=5. Then the weight (A, B) and cell numbers of thymus and spleen (C, D) were measured. Results are expressed as mean ± SD.

Figure 2. Chronic psychological stress induced the accumulation of CD11b+Gr1+ cells.

BALB/c mice were subjected to 5-days restraint stress or left untreated, n=5. Bone marrow cells (A-C) and peripheral blood cells (D-F) were subjected to flow cytometry for CD11b and Gr1 staining. (A, D) Representative data. (B, E) Percentages. (C, F) Absolute numbers. Results are expressed as mean ± SD.

Figure 3. Catecholamines collaborated with other factors to induce the accumulation of CD11b+Gr1+ cells.

BALB/c mice were subjected to 5-days restraint stress or left untreated, n=5. (A) One hour before each restraint session, mice were i.p injected with mifeprostone (RU486) and antalarmin (Anta), or same volume of solvent propylene glycol/ethanol. (B, C) One hour before each restraint session, mice were i.p injected with propranolol (Prop), epinephrine (EPI), or same volume of solvent PBS. (D) Mice were i.p injected with isoproterenol (ISO) or same volume of solvent PBS once daily for 5 days. Then, bone marrow cells were subjected to flow cytometry for CD11b and Gr1 staining. (A, B, D) Percentages. (C) Absolute number. Results are expressed as mean ± SD.

Figure 4. COX-2-PGE₂ loop mediated the accumulation of CD11b+Gr1+ cells.

BALB/c mice were subjected to 5-days restraint stress or left untreated. In some experiments, mice were i.p injected with SC-236 or same volume of solvent DMSO one hour before each restraint session. (A) Peripheral blood was sampled and serum PGE₂ and VEGF levels were analyzed by ELISA. Results are expressed as mean ± SD, n=5. (B) Bone marrow Gr1+ cells were purified and subjected to immunoblotting, n=3. One representative of two independent experiments with similar results is shown. Densitometric readings are shown for COX-2 and normalized with β-actin (set the lowest ratio as 1.0). (C) Bone marrow cells were subjected to flow cytometry for CD11b and Gr1 staining. Percentages of CD11b+Gr1+ cells were shown. Results are expressed as mean ± SD, n=5.

Figure 5. Accumulated CD11b+Gr1+ cells under the condition of chronic psychological stress were MDSCs.

BALB/c mice were subjected to 5-days restraint stress or left untreated. Bone marrow Gr1+ cells were purified. (A) Bone marrow-derived macrophages were co-cultured with bone marrow CD11b+Gr1+ cells at a ratio of 1:4, and then were stimulated with or without 50 ng/ml LPS for 24 hours. Cytokine release was determined by ELISA. Results are expressed as mean ± SD, n=5. (B) CD4+ CD25- splenic T cells were purified and labeled with CFSE. Then, T cells were co-cultured with bone marrow CD11b+Gr1+ cells at a ratio of 1:1 or 0.5:1. Stimulation was affected by antibodies against CD3 and CD28 for 96 hours. Proliferation was assessed by flow-cytometric analysis of CFSE dilution. One representative of two independent experiments with similar results is shown.

Figure 6. Accumulated MDSCs expressed multiple inhibitory molecules.

BALB/c mice were subjected to 5-days restraint stress or left untreated, n=5. (A) Bone marrow Gr1+ cells were purified and subjected to real time PCR, Results are expressed as mean ± SD. (B) Bone marrow cells were subjected to flow cytometry for ROS production assays, representative data are shown. (C) Bone marrow cells were subjected to flow cytometry for PD-L1, PD-L2 and FasL expression, representative data are shown.