Additional file 1 of Environmental signals perceived by the brain abate pro-metastatic monocytes by dampening glucocorticoids receptor signaling
Creators: name: Canali, María Magdalena
name: Guyot, Mélanie
name: Simon, Thomas
name: Daoudlarian, Douglas
name: Chabry, Joelle
name: Panzolini, Clara
name: Petit-Paitel, Agnès
name: Hypolite, Nicolas
name: Nicolas, Sarah
name: Bourdely, Pierre
name: Schmid-Antomarchi, Heidy
name: Schmid-Alliana, Annie
name: Soria, Javier
name: Karimdjee Soilihi, Babou
name: Hofman, Paul
name: Prevost-Blondel, Armelle
name: Kato, Masashi
name: Mougneau, Evelyne
name: Glaichenhaus, Nicolas
name: Blancou, Philippe
Publisher: name: figshare
Year: 2024
Subjects: subject: Medicine
subject: Genetics
subject: FOS: Biological sciences
subject: Physiology
subject: Immunology
subject: FOS: Clinical medicine
subject: Developmental Biology
subject: Cancer
subject: Mental Health
subject: Plant Biology

description: Additional file 1: Fig. S1. Behavior and adult hippocampal neurogenesis in SE and EE mice.SE and EE mice were analyzed using the openfield test (A), the light and dark paradigm test (B), the Novelty SuppressedFeeding (NSF) test, (C) the Forced Swimming Test (FST) (D), and the Barnes mazetest (E). (A) Time spent in the aversive center of the open field arena inseconds (left). Number of entries in the central area (right). (B) Time spentin light. (C) Latency to eat. (D) Immobility time. (E) Spatial learning curveestablished by training in the Barnes maze for four consecutive days. (F) Numberof BrdU-positive nuclei in the dentate gyrus three weeks after BrdU treatment.Each dot represents a mouse (A-D, F). Mean ± s.e.m. of two experiments. *, p &lt; 0.05; **, p &lt; 0.01; ****, p &lt; 0.0001. Fig. S2. Number of lymphoid and myeloid immune cell types in thesecondary lymphoid organs of SE and EE mice before tumor cell injection.C57BL/7 mice were housed under SE or EEconditions for 10-12 weeks and cell suspensions were prepared from the spleen(A, B) and mesenteric LN (C). (A) CD4+ T lymphocytes (CD4+CD3+), CD8+ T lymphocytes (CD8+ CD3+),B lymphocytes (CD19+ CD3-), NK T cells (NK1.1+CD3+), NK cells (NK1.1+ CD3-), neutrophils(CD11b+ CD11c- F4/80-Ly6C- Ly6Ghigh),CD11b+ DCs (CD11b+CD11c+), CD8+DCs (CD8+CD11c+), plasmacytoid DCs (CD11c+CD11b-120G8+),naïve CD4+ T cells (CD4+ CD3+ CD44low,CD62Lhigh), effector/memory CD4+ T cells (CD4+CD3+ CD44high) , regulatory CD4+ T cells (CD4+FoxP3+), naive CD8+ T cells (CD8+ CD3+CD44low, CD62Lhigh), memory CD8+ T cells (CD8+CD3+ CD44high) were identified by flow cytometry based onthe indicated surface markers. (A) Number of lymphoid cell types in spleennormalized to the mean values determined in SE mice. (B) Number of myeloid celltypes in spleen normalized to the mean values determined in SE mice. (C) Numberof lymphoid cell types in mesenteric LN normalized to the mean valuesdetermined in SE mice. Mean ±s.e.m. of two experiments. Fig. S3. Cytokine and chemokine levels and frequency of immune celltypes in the lungs of SE and EE mice before tumor cell injection.C57BL/7 mice were housed under SE or EEconditions for 10-12 weeks. (A, B) Lung protein extracts were prepared and thelevels of the indicated cytokines (A) and chemokines (B) were measured. (C, D)Lung cell suspensions were prepared and the number of NK cells (NK1.1+CD3-), T lymphocytes (CD19- CD3+), Blymphocytes (CD19+ CD3-), inflammatory monocytes(Siglec-F-CD11c- Ly6G-CD11bhighMHCII- CD64- Ly6C+), patrolling monocytes(Siglec-F-CD11c- Ly6G-CD11bhighMHCII-CD64-Ly6C-), neutrophils (Siglec-F-CD11c- CD103- CD11b+Ly6Ghigh),CD11b+ DCs (Siglec-F-CD11c+ CD11bhighMHCII+CD64- CD24+), CD103+ DCs(Siglec-F-CD11b-CD103+ CD11c+CD24+),interstitial macrophages (Siglec-F-CD11c- CD11bhighMHCII+CD64+ CD24-), alveolar macrophages(CD11b- Siglec-F+CD11c+ CD64+) wasdetermined by flow cytometry based on the indicated surface markers. (A)Cytokine levels in lung normalized to the mean values determined in SE mice.(B) Chemokine levels in lung normalized to the mean values determined in SEmice. (C) Number of myeloid cell types in lung normalized to the mean valuesdetermined in SE mice. (D) Number of lymphoid cell types in lung normalized tothe mean values determined in SE mice. Mean ± s.e.m. of two experiments. Fig. S4. Phenotypiccharacterization of LysM-Cre+:GRfl/fl and LysM-Cre+: Stopfl/+TdTomato  transgenic mice.    (A) Lungcells from LysM-Cre+:Stopfl/+TdTomato micewere analyzed by flow cytometry after gating on CD45+ cells and thefrequencies of TdTomato+ cells among alveolar macrophages (CD11blowCD11c+ CD64+ Ly6C-Ly6G- Siglec-F+),interstitial macrophages (CD11b+ CD11c+ CD24-Ly6C+ Ly6G- MHCII+Siglec-F-),neutrophils (CD11b+ CD11c- F4/80-Ly6C-Ly6Ghigh), inflammatory monocytes (CD11b+ CD11c-CD24-Ly6Chigh Ly6G-MHCII+/-) patrollingmonocytes (CD11b+CD11cmed CD24- Ly6ClowLy6G- MHCII- Siglec-F-), DCs (CD11c+Ly6C-Ly6G-), and T lymphocytes (CD3+), were determined.Frequency of TdTomato+cells among the indicated cell types in arepresentative mouse. (B) Peritoneal macrophages from LysM-Cre+:GRfl/flandGRloxP/loxP mice were incubated with LPS in the presence ofthe indicated concentrations of corticosterone. Cellular supernatants wereassessed for TNF-a secretion 24 hours later. Percentage ofinhibition of TNF-a secretion relative to cells incubated in theabsence of corticosterone. Mean ± s.e.m. of 4 mice/group. (C) Lung, spleen andblood cells from LysM-Cre+:Stopfl/+TdTomato and GRloxP/loxP. mice were analyzed by flow cytometry aftergating on CD45+ cells for all immune cells and CD45-cells for endothelial cells. Mean ±s.e.m. of 4 mice/group. Fig. S5. Behavioralcharacterization of LysM-Cre+:GRfl/fl .(A-D) GRloxP/loxP  and LysM-Cre+:GRfl/fl micewere housed for 10 weeks under SE or EE conditions and analyzed using the openfield test (A), the light and dark paradigm test (B), the Novelty SuppressedFeeding (NSF) test (C) and the forced swimming test (FST) (D). (A) Time spentin the aversive center of the open field arena. (B) Time spent in light. (C)Latency to eat. (D) Immobility time. Mean ±s.e.m.; **, p &lt; 0.01; ***, p &lt; 0.001; ****, p &lt; 0.0001. Fig. S6. Flow cytometry gating strategy used for analyzinglung-infiltrating immune cells and immunophenotyping of Ly6C-depletedmice. (A) Flowcytometry gating strategy. After isolation, lung cells were stained with 7-AADand mAbs to CD11b, CD11c, CD24, CD45, CD64, CD103, F4/80, Ly6C, Ly6G, MHCII,Siglec-F and analyzed by flow cytometry. The gating strategy for identifyingalveolar macrophages (AM)(CD11b- Siglec-F+CD11c+CD64+), CD103+ DCs (Siglec-F-CD11b-CD103+CD11c+CD24+), neutrophils (Siglec-F-CD11c-CD103- CD11b+ Ly6Ghigh), inflammatorymonocytes (iMo) (Siglec-F-CD11c- Ly6G-CD11bhighMHCII- CD64- Ly6C+), patrolling monocytes(pMo) (Siglec-F-CD11c- Ly6G-CD11bhighMHCII- CD64- Ly6C-), interstitial macrophages(IM) (Siglec-F-CD11c- CD11bhigh MHCII+CD64+CD24-) and CD11b+ DCs (Siglec-F-CD11c+CD11bhigh MHCII+CD64- CD24+) isshown. (B) Lung, slpeen and blood cells from Isotype-control and anti-Ly6C-treated mice were analyzed by flow cytometry aftergating either on CD45+ cells for all immune cells or CD45-cells for endothelial cells. It is to be noted that anti-Ly6C clone Monts-1 wasused as depleting antibody whereas anti-Ly6C clone AL-21 was used for flowcytometry staining. Mean ± s.e.m. of 6 mice/group. ***, p &lt; 0.001; n.s. notsignificant. Fig. S7. Flowcytometry analysis of Ly6C+ cells in lung. After isolation, lung cells from wt mice werestained with mAbs to CD103, CD11b, CD11c, CD24, CD45, Ly6C, Ly6G, MHCII andanalyzed by flow cytometry after gating on CD45+ Ly6C+alive cells. Representative FACS profiles and proportions of cells within the indicatedgates. Fig. S8. Cytokineserum levels 4 days after tumor cell injection.C57BL/7 mice were housed under SE or EEconditions for 10-12 weeks. (A) Mice were injected with B16:F10 cells into thetail vein and serum samples were analyzed 4 days later for IFN-g,IL-1b,IL-6, TNF-a, and IL-10 levels. (B) Mice were treated witheither an anti-Ly6C or and isotype control mAb and injected one day after withB16:F10 cells into the tail vein. Serum samples were analyzed 4 days later forIFN-g,IL-1b,IL-6, TNF-a, and IL-10 levels. Cytokine levels inindividual mice normalized to the mean values determined in SE mice. Mean ±s.e.m. of three (A) or two (B) experiments. *, p &lt; 0.05; **, p &lt; 0.01;***, p &lt; 0.001; ****, p &lt; 0.001. Fig. S9. Differential transcriptomic, cytokine and chemokine profiles of inflammatorymonocytes in EE and SE mice.SE and EE mice were injected with CMRA-labelledB16:F10 cells and analyzed 6 hours later. (A-C) Lung cells were stained withDAPI and antibodies to CD45, CD11b and Ly6C, and analyzed by flow cytometry (A,C) or confocal microscopy after sorting of CMRA+Ly6Chighcells(B). (A) Gating strategy. (B) Confocal analysis of representative CMRA+Ly6Chighcells purified from SE mice after staining with anti-CD45 mAb (green).Arrows indicate CMRA+ cytoplasmic vesicles (red). (C) Number of CMRA+inflammatory monocytes in individual SE and EE mice. Fig. S10. Percentage of CMRA+ cells in SE and EE mice.C57BL/7 mice were housed under SE or EEconditions for 10-12 weeks and injected with CMRA-labeled B16:F10 cells. Lungcell suspensions were prepared 16 hours later and the percentage of CMRA+cells was measured after gating on neutrophils (Siglec-F-CD11c-CD103- CD11b+ Ly6Ghigh), inflammatorymonocytes (Siglec-F-CD11c- Ly6G-CD11bhighMHCII- CD64- Ly6C+), patrolling monocytes(Siglec-F-CD11c-Ly6G-CD11bhighMHCII- CD64-Ly6C-), interstitial macrophages(Siglec-F-CD11c-CD11bhighMHCII+CD64+CD24-) and CD11b+ DCs (Siglec-F-CD11c+CD11bhigh MHCII+CD64- CD24+).Percentage of CMRA+ in individual mice normalized to the mean valuesdetermined in SE mice. Mean ± s.e.m. two experiments. Fig. S11. Differential transcriptomic profiles of inflammatory monocytes in EE and SEmice.SE and EE mice were injected with CMRA-labelledB16:F10 cells and analyzed 6 hours later. Lung cells were stained with DAPI andantibodies to CD45, CD11b and Ly6C. CMRA+and CMRA-Ly6Chighcells were sorted and analyzed by RNA sequencing. (A) Volcano plotanalysis of genes differentially expressed in CMRA+ and CMRA-Ly6C+ cells in SE (left) and EE (right) mice. (B) Scatter plotrepresentation showing, for each transcript, the ratio between expression levelin CMRA+ and CMRA- cells in EE mice (Y axis) and in SEmice (X axis). (C) Clustering heat map of 16 samples based on the 289 genesdifferentially expressed between CMRA+ and CMRA- cells inSE and EE mice 6 hours after tumor cell injection. Fig. S12. Transcriptomicanalysis of Ly6C+ cells from LysM-Cre+:GRfl/flmice.LysM-Cre+:GRfl/flmiceand GRloxP/loxP were housed for 10-12 weeks under SE or EEconditions, and injected with CMRA-loaded B16:F10 cells into the tail vein.Lung cell suspensions were prepared 4 days later, and Ly6C+ cellswere sorted by flow cytometry and analyzed by RNA-seq transcriptomic profiling.Expression levels of CCL3, CCL4, Fos, Rel and IL-1b inindividual mice. Mean ± s.e.m. of 8 mice. *, p &lt; 0.05; **, p &lt;0.01; ***, p &lt; 0.001. Fig. S13. Schematic representation of the impact of the housingenvironment on the immune system during the late stages of lung metastasis.(A) Schematic representation of the impact ofenriched environment on the hypothalamic-pituitary-adrenal axis. Sensory,cognitive and motor stimuli are integrated in the hypothalamus. Glucocorticoidproduction by adrenal glands is regulated by the adrenocorticotropic hormone(ACTH), which is secreted by the anterior pituitary gland in response tocorticotropin-releasing hormone (CRH) produced in the hypothalamus. In EE mice,the activity (and weight) of adrenal glands is decreased resulting in lowercorticosterone serum levels. (B, C) Schematic representation of theinteractions between immune and tumor cells in the lung microenvironment 0-6hours (B) and 3-4 days (C) after B16:F10 cell injection. (B) Circulating tumorcells (CTCs) are arrested in lung micro-vessels and escape from the blood byextravasation. CTC-derived materials are generated within minutes of CTC entry (29) and are captured by inflammatory monocytes inthe lung parenchyma. Inflammatory monocytes are activated and secretechemokines and cytokines. (C) Cytokines and chemokines secreted by inflammatorymonocytes promote the recruitment of both NK and T cells to the metastatic siteand either the killing of pioneer metastatic cells or the inhibition of theirdivision. In EE mice, reduced corticosterone levels result in an increased secretionof CCL20, CXCL2, CXCL10, IL-1b and TNF-a by inflammatory monocytes. The extravasationand activity of NK and T cells are enhanced resulting in increased tumor cellkilling. Red arrows indicate levels in EE mice relative to SE mice.Adrenocorticotropic hormone (ACTH); Corticotropic releasing hormone (CRH). Table S1. RNA-seq transcriptomic profiling of CMRA+ andCMRA- Ly6C+ cells.SE and EE mice injected with CMRA-labelledB16:F10 cells. CMRA+ and CMRA- Ly6C+ cellswere sorted 6 hours later and analyzed by RNA-seq transcriptomic profiling.List of genes differentially expressed between CMRA+ and CMRA-cells in SE and EE mice respectively. Log2-foldchanges and FDR are indicated. Table S2. Top canonical pathways of genes differentially expressedbetween CMRA+ and CMRA- Ly6C+ cells.Ingenuity pathway analysis of genesdifferentially expressed in CMRA+ and CMRA- Ly6C+cells. The names of the top canonical pathways are indicated as well as theproportions of genes defining the CMRA signature that belong to each pathway.The indicated p-values and z-scores refers to the statistical significance ofeach pathway