Chronic variable stress activates hematopoietic stem cells …

Clinical study.

The clinical study titled 'Effects of Socioenvironmental Stress on the Human Hematopoietic System' was an open, monocenter, single-arm study that enrolled medical residents working on the intensive care unit at University Hospital, Freiburg, Germany. This study was registered with the German Registry for Clinical Studies (DRKS00004821) and was approved by the Ethics Committee of Albert-Ludwigs-University Freiburg, Germany (No. 52/13). All residents working on the ICU were considered eligible to participate in the study. Exclusion criteria were smoking, any acute or chronic illness, regular intake of medication or failure to consent. Twenty-nine volunteers (23 male, 6 female, mean age 33.7 0.8 years) were enrolled after signing the informed consent form. Residents gave two blood samples (baseline and stress). The off-duty sample (baseline) was collected after 10 0.9 consecutive days off duty. The on-duty sample (stress) was collected after 7 0.3 consecutive days of ICU duty. A subcohort of participants completed the Perceived Stress Scale 10-item inventory5 before starting to work on the ICU (baseline), as well as after several weeks on duty (stress). Short-term perception for stress frequency and intensity was measured with visual analog scales (scale 010)6, which each participant completed at the time of the blood sampling. The mean circadian time difference between the baseline and the stress sample was 20 15.9 min. Blood samples were analyzed in a blinded fashion at the routine clinical laboratory of the University Hospital, Freiburg, Germany.

We used C57BL/6, CD45.1 (B6.SJL-Ptprca Pepcb/BoyJ), UBC-GFP (C57BL/6-Tg(UBC-GFP)30Scha/J), Apoe/ (B6.129P2-Apoetm1Unc/J), TH-Cre (B6.Cg-Tg(Th-Cre)1Tmd/J) and iDTR (C57BL/6-Gt(ROSA)26Sortm1(HBEGF)Awai/J) mice, all female and 1012 weeks of age (Jackson Laboratories, Bar Harbor, ME). Adrb3/ mice16 were donated by P. Frenette (Albert Einstein College of Medicine, New York, NY, USA) and B. Lowell (Beth Israel Deaconess Medical Center, Boston, MA, USA). Nestin-GFP mice41 were a gift from G. Enikolopov (Cold Spring Harbor Laboratory, NY). All procedures were approved by the Subcommittee on Animal Research Care at Massachusetts General Hospital. For each experiment, age-matched female littermates were randomly allocated to study groups. Animal studies were performed without blinding of the investigator.

Mice were exposed to socioenvironmental stressors7,8,9 for one or three weeks in C57BL/6 mice or six weeks in Apoe/ mice. Stress procedures were performed between 7 a.m. and 6 p.m. The following stressors were applied. For cage tilt, the cage was tilted at a 45 angle and kept in this position for six hours. For isolation, mice were individually housed in an area one-quarter of the original cage size (12 cm 8 cm) for four hours, followed by crowding, during which 10 animals were housed in one cage for two hours. Mice were monitored during the crowding procedure, and 'fighters' were separated. For damp bedding, water was added to the cage to moisten the bedding without generating large pools. Mice were kept for six hours with damp bedding. For rapid light-dark changes, using an automatic timer, the light was switched with an interval of seven minutes for two hours. For overnight illumination, mice were housed in a separate room with illumination from 7 p.m. to 7 a.m. All stressors were randomly shuffled in consecutive weeks. Efficacy of the chronic stress procedures was confirmed by measurement of blood corticosterone levels (Supplementary Fig. 12c).

Mice were irradiated using a split dose of 2 600 cGy with an interval of 3 h between doses. Animals were irradiated 12 h before bone marrow reconstitution.

For competitive bone marrow repopulation assays42, we co-transferred 2 106 whole bone marrow cells from CD45.1 mice after three weeks of stress or from nonstressed controls together with equal cell numbers of CD45.2 competitor cells from nonstressed wild-type mice into lethally irradiated UBC-GFP CD45.2 mice. Engraftment was assessed by comparing blood leukocyte chimerism for CD45.1 cells between groups after 2, 3 and 4 months. For limiting dilution experiments42, donor doses of 1.5 104, 6 104, 12.5 104 or 5 105 whole bone marrow cells from CD45.1 mice after three weeks of stress or from nonstressed controls were co-transferred with 5 105 CD45.2 competitor cells into lethally irradiated CD45.2 recipients. Engraftment was assessed after four months as at least >0.1% multilineage blood chimerism for B lymphocytes, T lymphocytes and myeloid lineage cells derived from donor bone marrow. Poisson's statistic was calculated using L-calc software (Stemcell Technologies) and ELDA software43. Bone marrow of two mice was pooled for each cell population.

To inhibit 3-adrenergic signaling, a specific antagonist for the 3-adrenergic receptor (SR 59230A, Sigma-Aldrich) was injected at 5 mg/kg body weight i.p. twice per day44. For inhibition of 2-adrenergic signaling, ICI118,551 hydrochloride (Sigma-Aldrich) was injected daily at a dose of 1 mg/kg body weight i.p. (ref. 18) for three weeks. The control groups received saline injections.

TH-Cre mice were cross-bred with iDTR mice. 1012 week old female TH-iDTR mice were intraperitoneally injected with 0.1 g/kg body weight diphtheria toxin (DT) on day 0 and day 3 after initiation of stress procedures18. Age-matched littermates (TH-Cre, iDTR or WT) that were also stressed and injected with DT served as controls.

Nonstressed mice and mice that had been stressed for three weeks were injected intravenously with 150 mg/kg body weight 5-FU (Sigma)45 on day 0. Mice were then followed over the course of 21 days, and the absolute number of blood leukocytes was measured after 7, 14 and 21 days. Stress exposure continued for the remaining 3 weeks after 5-FU exposure.

Flushed bone marrow was passed through a 40-m cell strainer and collected in PBS containing 0.5% BSA and 1% FBS (FACS buffer). Aortas were excised, minced and digested in collagenase I (450 U/ml), collagenase XI (125 U/ml), DNase I (60 U/ml) and hyaluronidase (60 U/ml) (all Sigma-Aldrich) at 37 C at 750 r.p.m. for 1 h. For sorting niche cells, bones were harvested from nestin-GFP mice. Bone marrow endothelial cells (ECs) and mesenchymal stem cells (MSCs) were obtained by flushing out bone marrow, which was then digested in 10 mg/ml collagenase type IV (Worthington) and 20 U/ml DNase I (Sigma)46. For obtaining bone osteoblastic lineage cells, we crushed bones, washed off residual bone marrow cells three times and then digested and incubated the bone fragments47,48.

For myeloid cells, cells were first stained with mouse hematopoietic lineage markers (1:600 dilution for all antibodies) including phycoerythrin (PE) anti-mouse antibodies directed against B220 (BD Bioscience, clone RA3-6B2), CD90 (BD Bioscience, clone 53-2.1), CD49b (BD Bioscience, clone DX5), NK1.1 (BD Bioscience, clone PK136) and Ter-119 (BD Bioscience, clone TER-119). This was followed by a second staining for CD45.2 (BD Bioscience, clone 104, 1:300), CD11b (BD Bioscience, clone M1/70, 1:600), CD115 (eBioscience, clone M1/70, 1:600), Ly6G (BD Bioscience, clone 1A8, 1:600), CD11c (eBioscience, clone HL3, 1:600), F4/80 (Biolegend, clone BM8, 1:600) and Ly6C (BD Bioscience, clone AL-21, 1:600). Neutrophils were identified as (CD90/B220/CD49b/NK1.1/Ter119)low(CD45.2/CD11b)highCD115lowLy6Ghigh. Monocytes were identified as (CD90/B220/CD49b/NK1.1/Ter119)lowCD11bhigh(F4/80/CD11c)lowLy-6Chigh/low or (CD45.2/CD11b)highLy6GlowCD115highLy-6Chigh/low. Macrophages were identified as (CD90/B220/CD49b/NK1.1/Ter119)lowCD11bhighLy6Clow/intLy6GlowF4/80high. For hematopoietic progenitor staining, we first incubated cells with biotin-conjugated anti-mouse antibodies (1:600 dilution for all antibodies) directed against B220 (eBioscience, clone RA3-6B2), CD11b (eBioscience, clone M1/70), CD11c (eBioscience, clone N418), NK1.1 (eBioscience, clone PK136), TER-119 (eBioscience, clone TER-119), Gr-1 (eBioscience, clone RB6-8C5), CD8a (eBioscience, clone 53-6.7), CD4 (eBioscience, clone GK1.5) and IL7R (eBioscience, clone A7R34) followed by pacific orangeconjugated streptavidin anti-biotin antibody. Then cells were stained with antibodies directed against c-Kit (BD Bioscience, clone 2B8, 1:600), Sca-1 (eBioscience, clone D7, 1:600), SLAM markers10 CD48 (eBioscience, clone HM48-1, 1:300) and CD150 (Biolegend, clone TC15-12F12.2, 1:300), CD34 (BD Bioscience, clone RAM34, 1:100), CD16/32 (BD Bioscience, clone 2.4G2, 1:600) and CD115 (eBioscience, clone AFS98, 1:600). LSKs were identified as (B220 CD11b CD11c NK1.1 Ter-119 Ly6G CD8a CD4 IL7R)lowc-KithighSca-1high. HSCs were identified as (B220 CD11b CD11c NK1.1 Ter-119 Ly6G CD8a CD4 IL7R)lowc-KithighSca-1highCD48lowCD150high. Granulocyte macrophage progenitors were defined as (B220 CD11b CD11c NK1.1 Ter-119 Ly6G CD8a CD4 IL7R)lowc-KithighSca-1low(CD34/CD16/32)highCD115int/low. Macrophage dendritic cell progenitors were defined as (B220 CD11b CD11c NK1.1 Ter-119 Ly6G CD8a CD4 IL7R)lowc-Kitint/highSca-1low(CD34/CD16/32)highCD115high. Common lymphoid progenitors were identified as (B220 CD11b CD11c NK1.1 Ter-119 Ly6G CD8a CD4)lowc-KitintSca-1intIL7Rhigh. For staining endothelial cells, we used ICAM-1 (Biolegend, clone Yn1/1.7.4, 1:300), ICAM-2 (Biolegend, clone 3C4, 1:300), VCAM-1 (Biolegend, clone 429, 1:300), E-selectin (CD62E) (BD Bioscience, clone 10E9.6, 1:100), P-selectin (CD62P) (BD Bioscience, clone RB40.34, 1:100), CD31 (Biolegend, clone 390, 1:600), CD107a (LAMP-1) (Biolegend, clone 1D4B, 1:600) and CD45.2 (Biolegend, clone 104, 1:300). Streptavidinpacific orange was used to label biotinylated antibodies. Endothelial cells were identified as CD45.2low, CD31high and CD107aintermed/high. For analysis of human monocyte subsets, cells were stained for HLA-DR (Biolegend, clone L243, 1:600), CD16 (Biolegend, clone 3G8, 1:600) and CD14 (Biolegend, clone HCD14, 1:600) after red blood cell lysis (RBC Lysis buffer, Biolegend). Monocytes were identified using forward and side scatter as well as HLA-DR. Within this population, frequencies of monocyte subsets CD14high, CD16high and CD14high/CD16high were quantified.

For BrdU pulse experiments, we used APC/FITC BrdU flow kits (BD Bioscience). One mg BrdU was injected i.p. 24 h before organ harvest. BrdU staining was performed according to the manufacturer's protocol. For BrdU application over 7 days, osmotic micropumps (Alzet) filled with 18mg BrdU were implanted. For the BrdU label-retaining pulse chase assay, BrdU was added to drinking water (1 mg/ml) for 17 days11.

After surface staining, intracellular staining was performed according to eBioscience's protocol: cells were fixed and permeabilized using the Foxp3/Transcription Factor Staining Buffer Set (eBioscience) and then stained for the nuclear antigen Ki-67 (eBioscience, clone SolA15). Cell cycle status was determined using 4,6-diamidino-2-phenylindole (DAPI, FxCycle Violet Stain, Life Technologies).

To isolate HSPCs, we used MACS depletion columns (Miltenyi) after incubation with a cocktail of biotin-labeled antibodies (as described in the flow cytometry section) followed by incubation with streptavidin-coated microbeads (Miltenyi). Next, cells were stained with c-Kit and Sca-1, and LSKs were FACS-sorted using a FACSAria II cell sorter (BD Biosystems). To purify niche cells from hematopoietic cells, we used MACS depletion columns after incubation with a cocktail of biotin-labeled antibodies as above followed by incubation with streptavidin-coated microbeads. Cells were then stained with CD45.2, Sca-1, CD31 and CD51 (Biolegend, clone RMV-7, 1:100). Endothelial cells were identified as LinlowCD45lowSca-1highCD31high. Bone marrow MSCs were identified as LinlowCD45lowCD31lowSca-1high/intermediate and GFP+. Osteoblasts were LinlowCD45lowSca-1lowCD31lowCD51high. For adoptive transfer of GFP+ neutrophils and Ly6Chigh monocytes, bone marrow cells were collected from UBC-GFP mice for purification of neutrophils and monocytes using MACS depletion columns after incubation with a cocktail of PElabeled antibodies including B220, CD90, CD49b, NK1.1 and Ter-119 followed by an incubation with PE-coated microbeads. Aortic endothelial cells were identified as CD45.2lowCD31highCD107aint/high and FACS-sorted using a FACSAria II cell sorter.

We injected 2 106 neutrophils together with 2 106 Ly6Chigh monocytes intravenously into nonstressed and stressed Apoe/ mice (the mice were stressed for 6 weeks, and the cells were injected 2 days before the end of the 6 weeks). Aortas were harvested 48 h later. The number of CD11bhighGFP+ cells within the aorta was quantified using flow cytometry.

Aortic roots were harvested and embedded to produce 6-m sections that were stained using an anti-CD11b (BD Biosciences, clone M1/70, 1:15 dilution) or anti-Ly6G (Biolegend, clone 1A8, 1:25 dilution) antibody followed with a biotinylated secondary antibody. For color development, we used the VECTA STAIN ABC kit (Vector Laboratories, Inc.) and AEC substrate (DakoCytomation). Necrotic core and fibrous cap thickness were assessed using Masson trichrome (Sigma) staining. Necrotic core was evaluated by measuring the total acellular area within each plaque. For fibrous cap thickness, three to five measurements representing the thinnest part of the fibrous cap were averaged for each plaque as previously described49. For tyrosine hydroxylase staining, femurs were harvested and fixed in 4% paraformaldehyde for 3 h and then decalcified in 0.375 M EDTA in PBS for 10 days before paraffin embedding. Sections were cut and stained with antityrosine hydroxylase antibody (Millipore, AB152, dilution 1:100) after deparaffinization and rehydration. Sections were scanned with NanoZoomer 2.0-RS (Hamamatsu) at 40 magnification and analyzed using IPLab (Scanalytics).

For intravital microscopy of hematopoietic progenitors in the bone marrow of the calvarium, LSKs were isolated from either wild-type C57BL/6 or C57BL/6-Tg(UBC-GFP)30Scha/J mice and labeled with the lipophilic membrane dye DiD (1,1-dioctadecyl-3,3,3,3-tetramethylindodicarbocyanine perchlorate, Invitrogen). 25,000 labeled LSKs were transferred i.v. into nonirradiated C57BL/6 recipient mice. For blood pool contrast, TRITCdextran (Sigma) was injected immediately before imaging. OsteoSense 750 (PerkinElmer) was injected i.v. 24 h before in vivo imaging to outline bone structures in the calvarium50. In vivo imaging was performed on days 1 and 7 after the adoptive cell transfer using an IV100 confocal microscope (Olympus)15. Three channels were recorded (DiD excitation/emission 644/665 nm, OsteoSense 750 excitation/emission 750/780 nm, TRITCDextran excitation/emission 557/576 nm) to generate z stacks of each location at 2-m steps. Image postprocessing was performed using Image J software. Mean DiD fluorescence intensity was measured for each labeled cell and then normalized to the background by calculating the target to background ratio.

Colony-forming unit (CFU) assays were performed using a semisolid cell culture medium (Methocult M3434, Stem Cell Technology) following the manufacturer's protocol. Bones were flushed with Iscove's Modified Dulbecco's Medium (Lonza) supplemented with 2% FCS. 2 104 bone marrow cells were plated on a 35-mm plate in duplicates and incubated for 7 days. Colonies were counted using a low magnification inverted microscope.

Blood pressure and heart rate were measured using a noninvasive tail-cuff system (Kent Scientific Corporation) according to the manufacturer's instructions. For each value, the mean of three consecutive measurements was used.

Messenger RNA (mRNA) was extracted from aortic arches or bone marrow using the RNeasy Mini Kit (Qiagen) or from FACS-sorted cells using the Arcturus PicoPure RNA Isolation Kit (Applied Biosystems) according to the manufacturers' protocol. One microgram of mRNA was transcribed to complementary DNA (cDNA) with the high capacity RNA to cDNA kit (Applied Biosystems). We used Taqman primers (Applied Biosystems). Results were expressed by Ct values normalized to the housekeeping gene Gapdh.

After six weeks of stress, FMT-CT imaging was performed and compared to nonstressed, age-matched Apoe/ controls. Pan-cathepsin protease sensor (Prosense-680, PerkinElmer, 5 nmol) was injected intravenously 24 h before the imaging as previously described51.

Blood corticosterone levels were measured by ELISA (Abcam). Serum was collected between 10 a.m. and 12 p.m. For measurements of noradrenaline in the bone marrow, a 2CAT (AN) Research ELISA (Labor Diagnostika Nord) was used. One femur was snap-frozen and immediately homogenized in a catecholamine stabilizing solution containing sodium metabisulfite (4 mM), EDTA (1 mM) and hydrochloric acid (0.01 N). Prior to the ELISA, the pH of the sample was adjusted to 7.5 using sodium hydroxide (1 N). ELISAs for CXCL12 (R&D), IFN- (PBL Biomedical Laboratories) and IFN- (R&D) in the bone marrow were performed using one femur and one tibia per mouse14. ELISAs were performed according to the manufacturers' instructions.

Statistical analyses were performed using GraphPad Prism software (GraphPad Software, Inc.). Results are depicted as mean standard error of mean if not stated otherwise. For a two-group comparison, a Student's t-test was applied if the pretest for normality (D'Agostino-Pearson normality test) was not rejected at the 0.05 significance level; otherwise, a Mann-Whitney U test for nonparametric data was used. For a comparison of more than two groups, an ANOVA test, followed by a Bonferroni test for multiple comparison, was applied. For analysis of clinical data, a Wilcoxon test for matched pairs was used. P values of <0.05 indicate statistical significance. No statistical method was used to predetermine sample size.

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Chronic variable stress activates hematopoietic stem cells ...