Social Stress Mobilizes Hematopoietic Stem Cells to Establish Persistent Splenic Myelopoiesis

Abstract

Psychosocial stress accelerates myelopoietic production of monocytes and neutrophils that contributes to a variety of health complications ranging from atherosclerosis to anxiety. Here, we show that social stress in mice mobilizes hematopoietic stem progenitor cells (HSPCs) from the bone marrow that enter circulation, engraft into the spleen, and establish a persistent extramedullary hematopoietic depot. These splenic progenitors actively proliferate and differentiate into multiple cell types, including monocytes, neutrophils, and erythrocytes. Splenic erythropoiesis partially abrogates stress-induced anemia. Repeated injection with isoprenaline induces progenitor mobilization to the spleen, identifying a key role for β-adrenergic signaling. Moreover, protracted splenic production of CD11b⁺ cells persists for at least 24 days after the cessation of social stress. Thus, chronic stress establishes a persistent extramedullary hematopoietic depot that can modify a wide range of chronic disease processes and alter homeostasis of the bi-directional regulatory axis between the nervous and immune systems.

Keywords: extramedullary hematopoiesis; hematopoiesis; hematopoietic stem cells; myelopoiesis; spleen; stress.

Figure 1.. Social Stress Enhanced Myelopoiesis at the Expense of Lymphopoiesis and Erythropoiesis

(A–F) Male C57BL/6 mice were exposed to 6 repeated cycles of social defeat stress (Stress) or left undisturbed as controls. BM, blood, and spleen samples were collected 0.5 days after the final cycle of stress. Representative bivariate dot plots show the labeling and gating strategies for (A and B) BM, (C and D) blood, and (E and F) spleen. (G–J) Ter119⁺ erythrocytes (Es), B220⁺ or CD3⁺ lymphocytes (Ls), CD11b⁺/Ly6G⁺ granulocytes (Gs), and CD11b⁺/Ly6C^hi monocytes (Mos) in (G and H) BM, (I) blood, and (J) spleen. (K) Representative picture of spleen size. (L) Spleen weight. Data from (A)–(F) and (H)–(L) derived from 1 experiment (n = 6). Data in (G) derived from 2 experiments (N = 12). Bars represent means ± SEMs. Means with asterisks are significantly different from control (p < 0.05).

Figure 2.. Social Stress Mobilized Hematopoietic Progenitors to the Blood and Spleen

Male C57BL/6 mice were exposed to 6 repeated cycles of social defeat stress (Stress) or left undisturbed as controls. BM, blood, and spleen samples were collected 0.5 days after the final cycle of stress. (A–G) Representative images of (A) phenotyped colony forming units (CFUs) of cell lineages: GEMM, E, GM, Ms, and G. Representative pictures of CFUs collected and quantification of phenotyped CFUs are shown from the (B and C) BM, (D and E) blood, and (F and G) spleen of control and stress mice. In a separate experiment, male C57BL/6 mice were exposed to 6 repeated cycles of social defeat stress (Stress) or left undisturbed as controls. Spleen samples were collected 0.5 days after the final cycle of stress. (H) Representative bivariate density plots show the labeling and gating strategy for detection of Lin⁻/cKit⁺/Sca1⁺ (LSK) cells in the spleen. (I) Number of LSK cells in the spleen. (J) Representative histogram of DAPI^hi S-G2-M phase LSK cells. (K) Number of proliferating S-G2-M phase LSK cells in the spleen. Bars represent means ± SEMs. Data from (A)–(G) derived from 1 experiment, with n = 4, and data from (H)–(K) derived from 1 experiment, with n = 6. Means with asterisks are significantly different from control (p < 0.05).

Figure 3.. Social Stress Increased Self-Renewing and Long-Lived HSPCs in the Spleen

(A) Schematic representation of experimental design. CD45.1⁺ splenocytes from control or stress mice were mixed 1:1 with GFP⁺ BM competitor cells and were i.v. injected into CD45.2 myoablated host mice. Engraftment of donor cells was assessed in cheek blood 1 and 4 months after i.v. transfer. (B) Representative bivariate density plots of GFP⁺ and CD45.1⁺ cells collected from cheek blood 1 month after i.v. transfer. (C) The ratioofCD45.1 ⁺ cells to GFP⁺ cells (%)for each cell type (B220, CD11b, CD3) detected in the cheek blood from CD45.2 host mice at 1 or 4 months after i.v. transfer. (D) Schematic representation of experimental design. Magnetic-activated cell sorting (MACS)-isolated GFP⁺Lin⁻ BM cells were i.v. transferred into control or stressed mice 0.5 days after stress. Recruitment of transferred GFP⁺/Lin⁻ cells to the spleen was assessed 1 hr later. (E) Bivariate density plots of GFP⁺Lin⁻ cells in the spleen. (F) Number of GFP⁺Lin⁻ cells that were recovered in spleen. RSD, repeated social defeat. Data from (A)–(C) are derived from 1 experiment (n = 6–7), and data from (D)–(F) are derived from 2 experiments (N = 6). Bars represent means ± SEMs. Means with asterisks are significantly different from control (p < 0.05).

Figure 4.. β-Adrenergic Signaling Enhanced Myelopoiesis and Increased HSPCs in the Blood and Spleen

Male C57BL/6 mice were injected i.p. twice daily with vehicle (Veh) or 10 mg/kg/day isoprenaline (Iso). BM, blood, and spleen samples were collected 0.5 days after the final injection. (A) Percentage of TER119⁺ E, B220⁺ or CD3⁺ L, CD11b⁺/Ly6G⁺ Gs, and CD11b⁺/Ly6C^hi Mos (M) in BM. (B–D) Spleen weight is shown (B). Quantification of CFU in (C) blood and (D) spleen are shown. Data from (A)–(D) derived from 2 experiments (N = 6). Bars represent means ± SEMs. Means with asterisks are significantly different from control (p < 0.05).

Figure 5.. Social Stress Enhanced Erythromyeloid Progenitor Proliferation in the Spleen

Male C57BL/6 mice were exposed to 6 repeated cycles of social defeat (stress) or left undisturbed as controls. EdU (50 mg/kg) was injected intraperitoneally (i.p.) 0.5 days after stress and the spleen was collected 30 min later. (A) Representative collages of spleen cross-sections for EdU (green) and DraQ5 nuclear (red) labeling. (B) Representative collages of spleen cross-sections for Ter119 (green) and DraQ nuclear (red) labeling. White boxes highlight magnified regions, which are shown at right. White scale bars, 1 mm. In a separate experiment, mice were subjected to stress, as above. The spleen was collected 0.5 or 18 hr after injection with BrdU (50 mg/kg). (C) Representative bivariate density plots of BrdU and DAPI labeling for cell-cycle analysis. (D) Percentage of nucleated Es (Ter119/ DAPI⁺) in the spleen. (E) Percentage of nucleated Mos (CD14/ DAPI⁺) in the spleen. Percentage of proliferating DAPI⁺ nucleated Es in the spleen (F) 0.5 hr and (G) 18 hr post-BrdU injection. Percentage of CD11b⁺/ BrdU⁺ cells in the spleen (H) 0.5 hr and (I) 18 hr post-BrdU injection. Data derived from 2 experiments (N = 7–8). Bars represent means ± SEMs. Means with asterisks are significantly different from control (p < 0.05).

Figure 6.. Persistence of Extramedullary Myelopoiesis 24 Days after Social Stress

(A) Male CX₃CR1(CreERT2)/ROSA26(STOP^flfl-tdTom) were exposed to 6 repeated cycles of social defeat stress (Stress) or left undisturbed as controls. Next, mice were injected with Tam (1 mg i.p.) 1 or 23 days later. At the 24-day endpoint, spleens were collected and tdTomato expression was determined in Mos. (B) Gating strategy for tdTomato⁺ Mos in the spleen. (C) Percentage of tdTomato+ Mos in the spleen 24 days later. (D–F) Quantification of CFUs in the (D) BM, (E) blood, and (F) spleen 24 days after social defeat. In a separate experiment, male C57BL/6 mice were subjected to 6 repeated cycles of social defeat (stress) or left undisturbed as controls. Next, mice were injected with BrdU 24 days after social defeat, and myeloid cell proliferation was determined in the spleen. (G) Representative BrdU (green) and CD11b (red) labeling in the spleen. (H) Number of CD11b⁺/BrdU⁺ cells per field of view in the spleen 24 days after stress. Data from (B) and (C) are derived from 1 experiment (n = 6), (D)–(F) from 1 experiment (n = 3), and (G) and (H) from 1 experiment (n = 6). Bars represent means ± SEMs. Means with asterisks are significantly different from control (p < 0.05).