1. Immunology and Inflammation
  2. Medicine

Inflammation rapidly recruits mammalian GMP and MDP from bone marrow into regional lymphatics

  1. Juana Serrano-Lopez
  2. Shailaja Hegde
  3. Sachin Kumar
  4. Josefina Serrano
  5. Jing Fang
  6. Ashley M Wellendorf
  7. Paul A Roche
  8. Yamileth Rangel
  9. Leolene J Carrington
  10. Hartmut Geiger
  11. H Leighton Grimes
  12. Sanjiv Luther
  13. Ivan Maillard
  14. Joaquin Sanchez-Garcia
  15. Daniel T Starczynowski
  16. Jose A Cancelas  Is a corresponding author
  1. Divisions of Experimental Hematology, Cincinnati Children’s Hospital Medical Center, University of Cincinnati College of Medicine, United States
  2. Hoxworth Blood Center, University of Cincinnati College of Medicine, United States
  3. Hematology Department, Reina Sofía University Hospital/Maimonides Biomedical Research Institute of Córdoba (IMIBIC)/University of Córdoba, Spain
  4. Center for Cancer Research, National Cancer Institute, United States
  5. Experimental Immunology Branch, National Cancer Institute, National Institutes of Health, United States
  6. University of Pennsylvania Perelman School of Medicine, United States
  7. Institute of Molecular Medicine, Ulm University, Germany
  8. Immunobiology, Department of Pediatrics, Cincinnati Children’s Hospital Medical Center, University of Cincinnati College of Medicine, United States
  9. Center for Immunity and Infection, Department of Biochemistry, University of Lausanne, Switzerland
  10. Department of Cancer Biology, University of Cincinnati, United States
https://doi.org/10.7554/eLife.66190
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Cite this article
  1. Juana Serrano-Lopez
  2. Shailaja Hegde
  3. Sachin Kumar
  4. Josefina Serrano
  5. Jing Fang
  6. Ashley M Wellendorf
  7. Paul A Roche
  8. Yamileth Rangel
  9. Leolene J Carrington
  10. Hartmut Geiger
  11. H Leighton Grimes
  12. Sanjiv Luther
  13. Ivan Maillard
  14. Joaquin Sanchez-Garcia
  15. Daniel T Starczynowski
  16. Jose A Cancelas
(2021)
Inflammation rapidly recruits mammalian GMP and MDP from bone marrow into regional lymphatics
eLife 10:e66190.
https://doi.org/10.7554/eLife.66190
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7 figures, 3 videos, 1 table and 2 additional files

Figures

Figure 1 with 2 supplements
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Inflammation induces early mobilization of hematopoietic stem cells and progenitors to lymph organs in humans and mice.

(A) Content of side population (SP)cells in human lymph node (LN) by flow cytometry. LN biopsies had been blindly identified histologically as lymphadenitis, subcategorized in follicular (FL, black circles, n = 21), granulomatous (GL, green circles, N = 7), and viral (VL, pink circles, n = 5) and lymphomas, subcategorized in Hodgkin’s lymphoma (HL, blue circles, n = 12) or non-Hodgkin’s lymphoma (NHL, orange circles, n = 19). (B) Strategy for lipopolysaccharide (LPS) administration and collection of tissues (blood, kidneys, liver, spleen, bone marrow (BM), lymph node (LN), and thoracic duct (TD)) at specific times. LPS or vehicle control PBS was administered at the early rest phase into C57Bl/6 (CD45.2+) mice, and tissue specimens were collected before (zeitgeber time [ZT]4), 1 hr (ZT5), 3 hr (ZT7), 6 hr (ZT10), or 12 hr (ZT16) later. (C) Myeloid colony-forming-cell unit (CFU-C) content in peripheral blood (PB) from C57Bl/6 mice pre-treated with PBS (black circles) or LPS (blue circles) at different circadian cycle times. (D) Absolute neutrophil count in PB from C57Bl/6 mice pre-treated with PBS (black circles) or LPS (blue circles) at different circadian cycle times. (E–J) Myeloid CFU-C content in organs at different circadian cycle times. CFU-C contained in kidneys (E), liver (F), spleen (G), BM (H), LN (I), and TD (J) in response to PBS (black circles) or LPS (blue squares) at different circadian cycle times (n = 3–4 mice per time point and treatment). ZT4 = 10 am (time of LPS administration). ZT7: 1 pm (3 hr post-administration of LPS). ZT10: 4 pm (6 hr post-administration of LPS). ZT16: 10 pm (12 hr post-administration of LPS). Results are shown as mean ± SD. *p<0.05, **p<0.01.

Figure 1—figure supplement 1
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Clonogenic and long-term multilineage potential of human and murine hematopoietic stem cells and progenitors (HSC/P) in lymph node (LN).

(A) Representative flow cytometry dot plots of side population (SP) cells from human LN biopsies diagnosed with lymphadenitis (left flow panel) and non-Hodgkin’s lymphoma (NHL, right flow panel). SP cells form a tail cluster on the left side. (B) Graph represents the percentage of SP cells in human LN biopsies categorized according to their anatomical location. Supra-diaphragmatic location (solid circles) included neck and axillary LN and infra-diaphragmatic location (open circles) included mesenteric and inguinal LN. (C) Representative colony-forming unit (CFU-C) micrographs in human LN diagnosed with follicular lymphadenitis. Scale bar: 100 µm. (D) Clonogenic potential of lymphadenitis-derived SP cells (upper left; with magnification of a CFU-GM in outlined inset) and fluorescence-activated cell sorter (FACS) analysis of LN SP-derived hematopoietic progenitors (upper right and bottom panels) maintained for 1 week in culture as previously described (Esplin et al., 2011). All SP-derived progenitors were positive for pan-leukocyte surface marker CD45 but had heterogenic expression for CD34 and CD133 surface markers. (E, F) Representative FACS profiles of murine bone marrow (BM) HSC/P in response to in vivo administration of PBS (left panels) or lipopolysaccharide (LPS) (right panels) at specified zeitgeber time (ZT). (G–N) Time response in the BM content of LSK (G), long-term HSC (LT-HSC) (H), short-term HSC (ST-HSC) (I), multipotential progenitors (MPP) (J), granulocyte-macrophage progenitors (GMPs) (K), common myeloid progenitors (CMP) (L), megakaryocyte-erythroid progenitor (MEP) (M), and LK cells (N) in response to PBS (black lines) or LPS (blue lines). Values represent mean ± SD of a minimum of four mice per group. *p<0.05, **p<0.01, ***p<0.001.

Figure 1—figure supplement 2
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Myeloid progenitor migration to lymph node (LN) in response to lipopolysaccharide (LPS) is independent of NF-κB activation.

(A) Schema for the competitive long-term reconstitution potential of LN cells (donor cells) from C57Bl/6 (CD45.2+) mice treated with PBS/LPS and harvested at zeitgeber time (ZT)7 (3 hr) into lethally irradiated CD45.1+ B6.SJLPtprca Pep3b/BoyJ recipient mice (n = 3 mice per group). (B–E) Competitive repopulating unit (CRU) assay of LN cells as assessed by flow cytometry of allotype CD45.2-expressing cells in transplanted mice followed for up to 16 weeks. (B) Overall CRU as gated on CD45.2+ cells in response to donor cells from mice pre-treated with PBS (black line) or LPS (blue line). (C–E) Myeloid cell (C), T-cell (D), and B-cell (E) contributing CRU in response to donor cells from mice pre-treated with PBS (solid bar) or LPS (blue mosaic bar). (F) Representative example of fluorescence-activated cell sorter analysis for side population (SP) cells (upper dot plots) and SP-LSK potential (lower dot plots) from PBS- and LPS-treated LN or bone marrow (BM) tissues at ZT7 (3 hr). Hoechst: Hoechst 33342 staining for blue (blue) and red (red) fluorescence emissions. (G, H) Donor-derived chimera of sorted LN- or BM-SP cells obtained from LPS-treated mice.

Figure 2
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Draining of bone marrow (BM)-derived lineage negative cells into lymphatics.

(A) Schema for adoptive transfer of BM-derived lineage negative cells (Lin-) labeled with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) dye into lymphatic endothelium reporter Lyve1eGFP mice. By 16 hr after cell transplantation, lipopolysaccharide (LPS) or PBS were administered into the Lyve1eGFP mice at zeitgeber time (ZT)4 (10 am; time of LPS administration). BM and lymph node (LN) cells were analyzed at ZT5.5 (11.30 am, 1.5 hr after LPS administration), ZT7 (1 pm, 3 hr after LPS administration), and ZT10 (4 pm, 6 hr after LPS administration) for labeled Lin- cells (Lin-/DiI+). (B) Frequency of Lin-/DiI+ homed to BM (solid bars) (mosaic bars) after PBS (black solid bar) or LPS (blue solid bar) administration at ZT7 (1 pm, 3 hr after LPS administration) and ZT10 (4 pm, 6 hr after LPS administration). (C) Representative 3D reconstruction images of the whole bone by two-photon microscopy showing lymphatic vessels (Lyve1) surface marker (red), nuclei with DAPI (blue), and cortical bone with second harmonic signal (SHG, light blue). The scale bars, 100 μm. (D, i–iv) Intravital two-photon microscopy imaging (IVM) of long bones from Lyve1eGFP mice showing lymphatic vessels (displayed in green) near to the surface of the bone (blue, detected by SHG signal) and homed Lin-/DiI+ cells (displayed in red). (i, iii) IVM of PBS specimen. (ii, iv) IVM of LPS specimen. (E, F) Analysis and quantification of the distance of homed Lin-/DiI+ (red) cells to Lyve1eGFP (green) cells after PBS/LPS administration at ZT5.5 (11.30 am, 1.5 hr after LPS administration) analyzed by Imaris 7.7.2 software. (G) Two-photon microscopy examples of images of longitudinal femoral sections stained with anti-Lyve1 antibody and DAPI, and analyzed for specific fluorescence signal and SHG for cortical bone. (H, I) Representative of 3D reconstitution images of PBS- and LPS-treated LN tissues (H) and cross-sections of LPS-treated LN (I) analyzed by confocal microscopy showing the location of mobilized Lin-/DiI+ cells (red; nucleus stained by DAPI in blue) in relation with Lyve1+ cells (green; nucleus stained by DAPI in blue). The Z-stack dimensions of upper panels were X = 1266.95 μm, Y = 1266.95 μm, and Z = 344 μm. Calibrate: XY = 2.47 μm and Z = 4 μm. Resolution: 512 × 512 × 86. The Z-stack dimensions of lower panels were: X = 1259.36 μm, Y = 1259.36 μm, and Z = 132 μm. Calibrate: XY = 2.46 μm and Z = 4 μm. Resolution of images was 512 × 512 × 86. (J) Absolute count of mobilized Lin-/DiI+ cells counted within LN at ZT7 (1 pm, 3 hr after LPS administration, solid bars) and ZT10 (4 pm, 6 hr after LPS administration, mosaic bars) after PBS/LPS administration. N = 4–14 LNs analyzed per time point in a minimum of three mice per group and/or time point. Graph data depict mean ± SD. *p<0.05, **p<0.01.

Figure 3 with 1 supplement
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Lymph node (LN)-mobilized granulocyte-macrophage progenitors (GMPs) preferentially differentiate into dendritic cells.

(A) Comparative quantification of the content of bipotent and unipotent myeloid progenitors in LN at zeitgeber time (ZT)7 (1 pm, 3 hr after lipopolysaccharide (LPS) administration) after PBS/LPS administration. (B) Schema of isolation and transfer of bone marrow (BM)-derived GMP from β-actin-GFP reporter mice (CD45.2+) into C57Bl/6 (CD45.2+) mice (2 × 105 GFP+-GMP cells/mouse, n = 3 mice per group). After BM homing (17 hr), mice were treated with single and low dose of LPS (5 mg/kg) at ZT4 (day 1) and 7 days later BM and LN tissues were analyzed for green fluorescent protein (GFP) expression in myeloid populations by flow cytometry. (C, D) Graphs represent the percentage of GFP+-GMP differentiated to granulocytes (solid bars, Gr1++CD11b+CD11c-), macrophages (left mosaic bars, Gr1dimCD11b+CD11cneg), and cDC (right mosaic bars, Gr1-CD11b+ CD11c+) 7 days post-transferring after PBS/LPS administration by flow cytometry into LN (C) and BM (D). (E) Fluorescence-activated cell sorter strategy for macrophage-dendritic progenitor (MDP) content in BM and LN tissues from CX3CR-1GFP reporter mice (4–5 mice per group). Phenotypically, MDPs are defined as lineage-negative with high expression of the chemokine receptor Cx3cr1, c-fms (CD115) and Flt3 (P2), and intermediate expression of c-Kit. (F, G) Graphs show absolute numbers of MDP present in BM (F) and LN (G) 3 hr later (ZT7 [3 hr]) after PBS (black circles) or LPS (blue squares) administration. Values represent mean ± SD. *p<0.05, **p<0.01.

Figure 3—figure supplement 1
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Lipopolysaccharide (LPS) induces rapid migration of macrophage-dendritic progenitors (MDPs) to regional lymph node (LN) chains.

(A) Flow cytometry analysis of green fluorescent protein (GFP) MDPs in bone marrow showing decreased MDPs upon LPS treatment. (B) Flow cytometry analysis of migrated GFP MDPs in LN (per regional group) showing increased GFP+MDPs in inguinal and popliteal LNs. Black bar: EGFP+; blue bar: EGFP-. Experiments were performed per triplicate. ***p<0.001.

Figure 4 with 2 supplements
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Traf6 is a key regulator for migration of bone marrow (BM)-derived myeloid progenitors to lymph nodes (LNs) in a non-cell-autonomous manner.

(A) Schema of full chimeric mice made by non-competitive transplantation of CD45.2+ Mx1Cre;WT and Mx1Cre;Traf6flox/flox BM cells into lethally irradiated CD45.1+ B6.SJLPtprca Pep3b/BoyJ. Six weeks later Traf6 gene were deleted by intraperitoneal injection of poly(I:C). 1 week later we performed PBS/lipopolysaccharide (LPS) injection early in the rest phase (zeitgeber time [ZT]4 [10 am, time of LPS administration]) and LN-contained myeloid progenitors at ZT7 (1 pm, 3 hr after LPS administration) was scored by colony-forming unit (CFU) assay. (B) Absolute number of CFU-GM present in LN from wild-type (WT) (solid bars) and Traf6∆/∆ (orange bars) full chimeric mice (n = 6–7 mice per group) after PBS (black and orange solid bars) or LPS (blue and mosaic bars) administration. (C–H) In vitro transwell migration assay for BM-derived LK cells. (C) Experimental design for migration of WT or Traf6∆/∆ low-density (LD) BM cells (CD45.2+) toward a WT microenvironment generated by BM (CD45.1+) in the presence of LPS for 4 hr. (D) Graph represents the percentage migrated LK from WT (blue solid bar) or Traf6∆/∆ (orange mosaic bar) low-density BM (LDBM) cells to the bottom as depicted in (C). (E) Experimental design for migration of WT LDBM cells (CD45.1+) toward a gradient generated by WT or Traf6∆/∆ (CD45.2+) BM or LN cells in the presence of LPS for 4 hr. (F) Graph represents the percentage of LDBM LK migrated to the BM bottom (solid bars) or LN bottom (mosaic bars) as schemed in (E). (G) Experimental design for WT LDBM cells (CD45.1+) migration toward gradient generated by WT or Traf6∆/∆ LN-derived T-cells (CD45.2+/CD3e+/CD11b-/B220-) or B-cells (CD45.2+/CD3e-CD11b-/B220+) or myeloid cells (CD45.2+/CD3e-/CD11b+/B220-) in the presence of LPS for 4 hr. (H) Graph represents the percentage of migrated LK LDBM to the WT LN bottom (blue mosaic bars) or Traf6∆/∆ LN bottom (orange mosaic bars) as schemed in (G). In all cases, LK cell migration was determined by CD45 allotype analysis using flow cytometry in triplicate. (I) Absolute number of CFU-GM present in LN from Lyz2Cre;WT (solid bars) and Lyz2Cre;Traf6flox/flox (mosaic bars) full chimeric mice after PBS/LPS administration at ZT7 (3 hr). (J) Graph represents cumulative survival of Lyz2Cre;WT (blue line) and Lyz2Cre;Traf6flox/flox (orange line) after 10 mg/kg of LPS. (J) Survival curve after 30 mg/kg of b.w. injection in Lyz2Cre;WT (blue line) or Lyz2Cre;Traf6flox/flox (orange line). Values are shown as mean ± SD of two independent experiments with a minimum of three mice or replicates per group and experiment. *p<0.05, **p<0.01.

Figure 4—figure supplement 1
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Myeloid progenitor migration to lymph node (LN) in response to lipopolysaccharide (LPS) is independent of NF-κB activation.

(A) Representative PCR amplifications show Traf6 deleted in circulating cells in four Mx1cre;Traf6flox/flox mice after poly(I:C) injections (10 mg/kg/2 days × five doses). (B) IFN-γ (Ifng) levels in the lower chamber at 4 hr after allowing transwell migration of wild-type (WT) bone marrow (BM) progenitors toward Wt or Traf6-deficient BM or LN cells (from Figure 4E, F). (C–H) Cytokine levels in the lower chamber at 4 hr after allowing transwell migration of LK cell progenitors contained in WT low-density BM (LDBM) toward WT or Traf6-deficient LN cells (from Figure 4E, F). (C) IL1-α (Il-1α). (D) IL-2 (Il-2). (E) IL-13 (Il-13). (F) IL-4 (Il-4). (G) TNF-α (Tnf-α). (H) IL-10 (Il-10). (I–L) Analysis of LK progenitor mitration toward BM-derived macrophages with exogenous expression of IκBα mutant resistant to proteasome degradation. BM Lin- cells were transduced with a MSCV-puro-eGFP bicistronic retroviral vector encoding full length of IκBα mutant and GFP+ protein. EGFP+ Lin- cells were sorted and differentiated in culture by macrophage colony-stimulating factor (M-CSF) cytokine to macrophages. Green fluorescent CD11b+-macrophages were layered on bottom chamber and stimulated them with LPS to generate myeloid chemotaxis gradient. (I) Experimental schema for the analysis of LK (Lin-/c-Kit+/Sca1-) cell migration contained in LDBM from the upper chamber to the transduced macrophage bottom stimulated with LPS and LPS + monensin (LPS + Mon) for 4 hr as depicted in (K). (J) Example of gating strategy for the sorting of transduced macrophages used in the bottom chamber of transwell assays. (K) Representative fluorescence-activated cell sorter dot plots demonstrating gating strategy to identify migrating granulocyte-macrophage progenitor (GMP) populations from the transwell migration assays. (L) Graph represents LK cell migration toward transduced macrophages in the presence of LPS (solid bars) or LPS + Mon (mosaic bars). Values represent mean ± SD of three mice per group and experiment. Experiments were performed per triplicate. ND: not detectable; NS: not significant. *p<0.05, ***p<0.001.

Figure 4—figure supplement 2
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Inflammation induces temporal changes in chemokine and cytokine signatures in bone marrow (BM) and lymph node (LN).

(A) Cxcl12 in femoral or LN extracellular fluid and plasma after PBS/lipopolysaccharide (LPS) in vivo administration at zeitgeber time (ZT)7 (1 pm, 3 hr after LPS administration). (B) Heat map showing cytokine profiling release into the extracellular fluid of femora and LN in response to PBS/LPS at ZT5 (11 am, 1 hr after LPS administration) and ZT7 (1 pm, 3 hr after LPS administration). (C–N) Graphs represent levels of relevant cytokines and chemokines associated with migration/inflammatory response and released into LN extracellular fluid (black and green bars) or into femoral extracellular fluid (black and orange bars) after PBS/LPS administration into C57Bl/6 mice at ZT5 (11 am, 1 hr after LPS administration) and ZT7 (1 pm, 3 hr after LPS administration). (C–J) Extracellular LN levels of Gm-csf (C), G-csf (D), M-Csf (E), Mcp-1 (F), Ccl5 (G), eotaxin/Ccl11 (H), IL-13 (I), and IL-5 (J). (K–N) Extracellular BM levels of Ccl3 (K), Ccl4 (L), Tnfα (M), and IL-1α (N). Values are mean ± SE of two mice per treatment and experiment, pooled from two independent experiments. *p<0.05, **p<0.01, ***p<0.001; ****p<0.0001.

Figure 5 with 1 supplement
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Granulocyte-macrophage progenitor (GMP) cells drain into local lymphatics and not blood circulation in early inflammation via Ccl19/Ccr7.

(A) Graph represents soluble Ccl19 chemokine in femoral or lymph node (LN) extracellular fluid and peripheral blood (PB) plasma after PBS (black lines) or lipopolysaccharide (LPS) (blue lines) administration at different circadian cycle times (zeitgeber time [ZT]4.5 [10.30 am, 0.5 hr after LPS administration], ZT5 [11 am, 1 hr after LPS administration], and ZT7 [1 pm, 3 hr after LPS administration]). (B) Strategy for in vivo neutralization of Ccr7 receptor or Ccl19 ligand by injections of anti-Ccr7 antibody or anti-Ccl19 antibody (50 μg/dose, two doses) into C57Bl/6 mice. One day after the last dose of antibodies, PBS or LPS was administered at ZT4 (10 am, time of LPS administration), and the myeloid progenitors-circulating cells from the LN and PB were measured by colony-forming unit (CFU) assay at ZT7 (1 pm, 3 hr after LPS administration). (C, D) Absolute number of progenitors present into LN from neutralized mice with anti-Ccl19/IgG (left graph) or anti-Ccr7/IgG2a (right graph) after PBS (black) or LPS (blue) administration as depicted in (B). (E) Generation of hematopoietic chimeric Ccl19 expressing (wild-type [WT]) or not (Ccl19-/-) mice and isolation of LNs after administration of PBS or LPS. (F) CFU content of LN from either WT or Ccl19-/- hematopoietic chimeric animals treated with PBS or LPS. (G) Experimental design to analyze L-selectin dependence of femoral GMP migration to regional (or distant) LN after LPS administration. (H, I) Percentages of GFP+ cells in LN after administration of an isotype control or anti-L-selectin antibodies. (H) Frequency of GFP +GMP cells in regional LN after administration of LPS was not modified by L-selectin blockade in vivo. (I) Inhibition of the migration of GFP+ B-lymphocytes to regional LNs in mice pre-treated with anti-L-selectin antibody. In (B) and (C), LN were collected at ZT7 or 3 hr after LPS. Values represent mean ± SD of replicates in two or three independent experiments. *p<0.05, **p<0.01, ***p<0.001.

Figure 5—figure supplement 1
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Myeloid expression of Ccl19 ligand in lymph node (LN) and short-term differentiation of pre-treated granulocyte-macrophage progenitors (GMPs).

(A) Ccl21 in femoral or LN extracellular fluid and peripheral blood (PB) plasma after PBS (left lines) or lipopolysaccharide (LPS) (right lines) administration at different circadian cycle times. (B) Ccl19 released into the supernatant from sorted wild-type (WT) and Traf6-deficient LN T+ cells, B+ cells, and CD11b+ myeloid cells after LPS stimulation in vitro. (C–E) Experimental schema of analysis of regional migration from chimeric femora of Ccr7-expressing GMP cells to bone marrow (BM) (D) and regional (inguinal and popliteal) LN (E). (F, G) Representative example of flow cytometry analysis (F) and mean fluorescence intensity (MFI) levels (G) of Ccr7 on GMPs from wild-type (WT) and Traf6-deficient mice in the presence of PBS (black and orange solid bars) or LPS (blue solid bar and orange mosaic bar). Values represent mean ± SD of two independent experiments. **p<0.01, ***p<0.001.

Figure 6 with 1 supplement
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In vivo analysis of Ccl19/Ccr7 axis during inflammation.

Pharmacological regulation of lipopolysaccharide (LPS)/Toll-like receptor (TLR) signaling pathway. (A) Annexin-V binding to membrane phosphatidylserine (PS) on lymph node (LN) myeloid populations from wild-type (WT) (left mosaic bars) and Traf6∆∕∆ (right mosaic bars) (n = 4 mice per group) after PBS or LPS administration. LN suspension cells were stained for myeloid surface markers including annexin-V and analyzed by flow cytometry. (B) Transwell migration of low-density bone marrow (LDBM)-derived LK cells toward gradient generated by pre-treated LN cells with dimethylsulfoxide (DMSO) (solid bars) as vehicle control and inhibitors (mosaic bars) against Irak1/4 (right lined), Ubc13 (left lined), and IKK (white squares), and following TLR signaling pathway activation by PBS (black) or LPS (blue). (C) Analysis of SNAP23 phosphorylation (Ser95) in LN myeloid cells previously treated with DMSO (solid bar) as vehicle control, Irak1/4 (right lined mosaic bar), Ubc13 (left lined mosaic bar), or IKK (white squares mosaic bar) inhibitors. Values represent two independent experiments as mean ± SD of two independent experiments performed in triplicate. (D) Mean fluorescence intensity (MFI) quantification of pre-stored Ccl19 into LN-residing CD11blow/CD11c+ cDC from non-manipulated mice by flow cytometry. Values represent mean ± SD of two or three independent experiments. p<0.05, **p<0.01, ***p<0.001.

Figure 6—figure supplement 1
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Traf6 does not associate with changes in survival of hematopoietic cells, and progenitors and Ccl19 can be detected in a specific subpopulation of lymph node (LN) conventional dendritic cells (cDCs).

(A) Apoptosis (TUNELassay) of LK progenitors, Lin- and Lin + bone marrow (BM) cells. (B) Representative overlap histograms of phospho-SNAP23 (pSNAP23) into LN myeloid cells treated with dimethylsulfoxide (DMSO) (upper left), Irak1/4-inh (upper right), Ubc13-inh (lower left), and IKK-inh (lower right) and stimulated with PBS (red lines) or lipopolysaccharide (LPS) (blue lines). (C) Gating strategy to determine Ccl19 pre-stored into LN-residing myeloid cells basally. (D) Mean fluorescence intensity (MFI) quantification of pre-stored Ccl19 in pDC populations in basal conditions.

Author response image 1
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Peripheral blood count of myeloid progenitors at ZT7 (3 hours after PBS or LPS administration).

A. Counts in Wt vs Traf6-deficient mice. B. Counts in Wt mice treated with either an isotype control (IgG2a) or neutralizing anti-Ccr7 antibody. C. Counts in Wt mice treated with an isotype control (IgG2a) or anti-Ccl19 neutralizing antibody. No difference is statistically significant.

Videos

Video 1
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Representative example of multiphoton microscopy processed with Imaris software of a femur from a Lyve1eGFP mouse treated with PBS at 1 hr after administration (11 am, 1 hr after lipopolysaccharide administration).

Bone tissue is identified as second-harmonic signal (blue). Imaris software was used to measure distance between DiI-labeled cells and green fluorescent protein-positive lymphatic vessels using 3D images.

Video 2
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Representative example of multiphoton microscopy processed with Imaris software of a femur from a Lyve1eGFP mouse treated with LPS at 1 hr after administration (zeitgeber time 5).

Bone tissue is identified as second-harmonic signal (blue). Imaris software was used to measure distance between DiI-labeled cells and green fluorescent protein-positive lymphatic vessels using 3D images.

Video 3
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Representative example of transcortical lymphatics discovered by staining with anti-Lyve1 (red) in wild-type C57Bl/6 mice.

Bone tissue is identified as second-harmonic signal (blue). Green signal is autofluorescence.

Tables

Appendix 1—key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional
information
Genetic reagent
(Mus musculus)		C57Bl/6Harlan Labs.
044		RRID:MGI:2161078C57BL/6NHsd; CD45.2+
Genetic reagent
(M. musculus)		B6.SJLPtprca Pepcb/BoyJJackson Labs 002014RRID:MGI:109863CD45.1+
Genetic reagent
(M. musculus)		Mx1-CreJackson Labs
003556		RRID:MGI:4358794Tg(Mx1-cre)1Cgn
Genetic reagent
(M. musculus)		Traf6flox/floxPMID:29386112Provided by Dr. Daniel Starczynowski, CCHMC, Cincinnati, OH
Genetic reagent
(M. musculus)		Lyz2CrePMID:10621974RRID:MGI:1934631Mature myeloid cell promoter driving Cre recombinase expression
Genetic reagent
(M. musculus)		Vav-CrePMID:14975238Provided by Dr. Andrew Roberts, WEHI, Melbourne, Australia
Genetic reagent
(M. musculus)		Lyve1eGFPJackson Labs 012601RRID:MGI:4421655Lyve1tm1.1(EGFP/cre)Cys
Enhanced green fluorescent protein (EGFP) in these knock-in mice is driven by the Lyve1 (lymphatic vessel endothelial hyaluronan receptor 1) promoter
Genetic reagent
(M. musculus)		β-actin-eGFPJackson Labs, 003291RRID:MGI:2686773B6 ATCTb-EGFP
Enhanced GFP (eGFP) cDNA under the control of a chicken β-actin promoter and cytomegalovirus enhancer, have widespread eGFP fluorescence, with the exception of erythrocytes and hair
Genetic reagent
(M. musculus)		CX3CR-1GFPJackson Labs
005582		RRID:MGI:J:84544Enhanced green fluorescent protein (EGFP) sequence replacing the first 390 bp of the coding exon (exon 2) of the chemokine (C-X3-C motif) receptor 1 (Cx3cr1) gene
LipopolysaccharideLPSSigma-AldrichCat# L2630Biological source is Escherichia coli (O111:B4)
Purified by phenol extraction
Stimulation with this generates various pro inflammatory cytokines
EnzymeCollagenase-IIThermo Fisher Scientific, GibcoCat# 17101015Biological source is Clostridium histolyticum, digestion of bone, cartilage, tissues
EnzymeDispaseThermo Fisher Scientific, Gibco.Cat# 17105041Biological source is Bacillus polymyxa
Helpful in dissociation of primary cells from the tissues
Lysing bufferPharm Lyse (red blood
lysing buffer)		BD BiosciencesCat# 555899Helpful in red blood cell lysis and results in good light scatter separation of lymphocytes and red blood cell debris when used in flow cytometry
Methylcellulose mediaMethocult-GF M3434StemCell TechnologyCat# 03434Semisolid medium to growth murine HPCs
Base methylcellulose mediumMethocult-M3134StemCell TechnologyCat# 03134Incomplete medium that contains 2.6% methylcellulose in Iscove's MDM
CytokineRecombinant murine
GM-CSF		PeproTechCat# 315-03Biological source is E. coli
Hematopoietic growth factor stimulates macrophages and neutrophils
Produced in endothelial cells, fibroblasts
CytokineRecombinant
murine G-CSF		PeproTechCat# 250-05Biological source is E. coli
Hematopoietic growth factor that stimulates the committed progenitor to neutrophils and improve the functional activities
CytokineRecombinant murine M-CSFPeproTechCat# 315-02Biological source is E. coli
It facilitates the hematopoietic recovery after the bone marrow transplantation
CytokineHuman G-CSFNEUPOGEN
(filgrastim, Amgen)
Lineage cocktail antibodyAnti-CD3e (CD3ε chain), anti-TER-119/erythroid cells (Ly-76), anti-Gr1 (Ly6G and Ly-6C), anti-CD45R (B220), anti-CD11b (integrin α chain, Mac1α)Pharmingen,
BD Biosciences		559971
RRID:AB_10053179		Sorting out or depleting lineage expressing cells and enriching hematopoietic progenitors in bone marrow
CD3e is biotin Hamster anti-mouse, dilution 1:200, CD45R is rat anti-mouse dilution 1:200, CD11b is rat anti-mouse, Ter119 is rat anti-mouse
Recombinant proteinCy7-APC-Cy7-conjugated streptavidinPharmingen,
BD Biosciences		554063
RRID:AB_10054651
AntibodyAPC-conjugated anti-c-KitPharmingen,
BD Biosciences		553356
RRID:AB_398536		Clone 2B8
Rat anti-mouse specifically binds to C-kit a transmembrane tyrosine kinase receptor
Dilution 1:200
AntibodyPECy7-conjugated anti-Sca1Pharmingen,
BD Biosciences		558162
RRID:AB_647253		Clone D7
Rat anti-mouse

Dilution 1:200
AntibodyeFluor 450-conjugated anti-CD34Affymetrix, eBioscienceRRID:AB_2043838Clone RAM34
Monoclonal antibody reacts with mouse CD34
Dilution 1:200
AntibodyPerCP Cy5.5-conjugated anti-Fcγ-RII/IIIPharmingen,
BD Biosciences		560540
RRID:AB_1645259		Clone 2.4G2
It specifically recognizes a common non-polymorphic epitope on the extracellular domains of the mouse FcrγIII Rat anti-mouse
Dilution 1:200
AntibodyFITC-conjugated anti-CD45.1Pharmingen,
BD Biosciences		553775
RRID:AB_395043		Clone A20
Monoclonal antibody specifically binds to CD45.1 of all leukocytes
Dilution 1:200
AntibodyPECy7-conjugated anti-CD45.2Pharmingen,
BD Biosciences		560696
RRID:AB_1727494		Clone 104
This recognizes CD45 on all leukocytes that of most mouse strains example: C57BL6, Balb/C, etc.
Mouse monoclonal
Dilution 1:200
AntibodyAPC-conjugated anti-CD11bPharmingen,
BD Biosciences		Cat# 553312
RRID:AB_398535		Clone M1/7
Specifically binds to CD11b or integrin alfa M
Rat anti-mouse
Dilution 1:200
Antibody-APC-Cy7-conjugated anti-B220 (clone RA3-6B2Pharmingen,
BD Biosciences		552094
RRID:AB_394335		Clone RA3-6B2
Binds to an extracellular domain of the transmembrane CD45, which is expressed in all B lymphocytes
Rat anti-mouse
Dilution 1:200
AntibodyPE-conjugated anti-CD3ePharmingen,
BD Biosciences		552774
RRID:AB_394460		Clone 145-2 C11
Binds to T-cell receptor expressed CD3 complex that is expressed in thymocytes and mature T cells, etc.
Hamster anti-mouse
Dilution 1:200
AntibodyBD Horizon V450-conjugated anti-Gr1Pharmingen,
BD Biosciences		560454 RRID:AB_1645285RB6-8C5
Rat anti-mouse, clonality unknown
Dilution 1:200
AntibodyPerCP efluor710 anti-CD115Thermo Fisher Scientific, eBioscience46-1152-82
RRID:AB10597740		Clone AFS98
Monoclonal antibody against mouse Cd115, receptor of macrophage colony-stimulating factor
Dilution 1:200
AntibodyR-phycoerythrin conjugated anti-CD135BD BiosciencesCat# 562537
RRID:AB_2737639		A2F10.1
This is a monoclonal antibody specifically binds to Flk2-Flt3
Dilution 1:200
DNA dieHoechst 33342Fisher Scientific, InvitrogenCat# H3570This is a cell permeant nuclear counterstain that emits blue florescence when bound to dsDNA
10 mg/mL
AntibodyRabbit non-conjugated monoclonal anti-phospho-SNAP23(Ser95)Paul A. RocheCenter for Cancer Research, National Cancer Institute, NIH
Fixation bufferBD CytofixBD BiosciencesCat# 554655For immunofluorescent staining of intracellular cytokines
Fixation/permeabilization Kit
BD Cytofix/CytopermBD BiosciencesCat# 554714This provides fixation and permeabilization of the cells
Perm/Wash bufferBD Perm/WashBD BiosciencesCat# 554723This is used in intracellular cytokine staining to permeabilize cells and can be used as antibody diluent
AntibodySecondary Alexa Fluor 488-conjugated, goat anti-rabbitThermo Fisher Scientific,RRID:AB_2633280Used as secondary antibody
Goat polyclonal
Dilution 1:500
Software, algorithmFlowJo xV0.7https://www.flowjo.com/solutions/flowjoRRID:SCR_008520
In Situ Cell Death Detection KitTMR redSigma-Aldrich,
Roche		Cat# 12156792910This is used to detect apoptosis on a single-cell level
Depletion kitLineage Cell DepletionMiltenyi BiotechCat# 130-090-858Depletion of mature hematopoietic cells
Plasma membrane dyeDil stainThermo Fisher Scientific, InvitrogenCat# D3911This is a lipophilic membrane stain that diffuses laterally to stain the entire cell
Detergent
solution		Triton X-100Sigma-AldrichCat# X100A mild detergent used to break the cell membrane
AntibodyAnti-GFPAbcamCat# ab13970
RRID:AB_300798		Chicken polyclonal antibody
Dilution 1:500
AntibodyPurified rat anti-mouse panendothelial cell antigen550563
RRID:AB_393754		Clone Meca-32
Mouse monoclonal
Dilution 1:500
AntibodySecondary antibodies
goat anti-rat Alexa Fluor-488		Thermo Fisher Scientific, InvitrogenCat# A-11006
RRID:AB_2534074		Goat anti-rat
Clonality unknown
Dilution 1:500
AntibodySecondary antibodies goat anti-chicken Alexa Fluor-568Thermo Fisher Scientific, InvitrogenCat# A-11041
RRID:AB_2534098		Goat anti-chicken
Clonality unknown
Dilution 1:500
SoftwareImarishttp://www.bitplane.com/imaris/imarisRRID:SCR_007370
AntibodyAnti-mouse CD62LBioXcellCat# BE0021
RRID:AB_1107665		This antibody reacts with L-selectin that is expressed by neutrophils, monocytes, and the majority of T- and B cells-
Mouse monoclonal
Dilution 1:500
AntibodyAnti-CD19BD BiosciencesCat# 552854
RRID:AB_394495		Rat monoclonal antibody
Dilution 1:200
Enzyme-free cell dissociation bufferCell dissociation bufferThermo Fisher–GibcoCat# 13150016Dissociation of mammalian cells
Recombinant human fibronectin fragmentRetroNectinTakaraCat# T202This reagent promotes co-localization of lenti or retrovirus with the target cells and promotes the transduction efficiency
AntibodyAnti-CD169eBioscienceCat# 12-5755-82
RRID:AB_2572625		This antibody specifically binds to CD169, the receptor expressed in subset of macrophages and plays an important role in cell-cell adhesion
AntibodyAnti-F4/80Thermo Fisher Scientific, eBioscienceCat# 48-4801-82
RRID:AB_1548747		Rat anti-mouse
Dilution 1:200
Protease inhibitor cocktailComplete Protease inhibitor cocktailSigma-Aldrich,
Roche		Cat# 11697498001Inhibits broad spectrum of proteases
ELISAMouse Ccl19/MIP-3R&D SystemCat# DY440
ELISAMouse Ccl21/6KineR&D SystemCat# AF457
Recombinant proteinMouse anti-Ccr7R&D SystemCat# MAB3477
Recombinant proteinMouse anti-Ccl19R&D SystemCat# 440-M3
InhibitorUbiquitin-conjugating enzyme E2 N (UBE2N) inhibitor or Ubc13Sigma-AldrichCat# 662107This inhibitor controls the biological activity of UbcH13
InhibitorIRAK1 inhibitorSigma-AldrichCat# I5409
InhibitorPS1145 dihydrochlorideSigma-AldrichCat# P6624IκB kinase inhibitor
InhibitorMonensin Solution 100×Thermo Fisher Scientific, eBioscienceCat# 00-4505-51Inhibitor of intracellular transport
AssayTUNEL-TMRRocheCat# 12156792910Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL
) assay to detect apoptotic cells labeled with tetra-methyl-rhodamine

Additional files

Supplementary file 1

Histological categorization of human lymph node biopsies and their side population (%) frequency.

https://cdn.elifesciences.org/articles/66190/elife-66190-supp1-v2.docx
Transparent reporting form
https://cdn.elifesciences.org/articles/66190/elife-66190-transrepform-v2.docx

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  1. Juana Serrano-Lopez
  2. Shailaja Hegde
  3. Sachin Kumar
  4. Josefina Serrano
  5. Jing Fang
  6. Ashley M Wellendorf
  7. Paul A Roche
  8. Yamileth Rangel
  9. Leolene J Carrington
  10. Hartmut Geiger
  11. H Leighton Grimes
  12. Sanjiv Luther
  13. Ivan Maillard
  14. Joaquin Sanchez-Garcia
  15. Daniel T Starczynowski
  16. Jose A Cancelas
(2021)
Inflammation rapidly recruits mammalian GMP and MDP from bone marrow into regional lymphatics
eLife 10:e66190.
https://doi.org/10.7554/eLife.66190