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Cutting Edge: LPS-Induced Emergency Myelopoiesis Depends on TLR4-Expressing Nonhematopoietic Cells

This information is current as Steffen Boettcher, Patrick Ziegler, Michael A. Schmid, of September 28, 2021. Hitoshi Takizawa, Nico van Rooijen, Manfred Kopf, Mathias Heikenwalder and Markus G. Manz J Immunol 2012; 188:5824-5828; Prepublished online 14 May 2012;

doi: 10.4049/jimmunol.1103253 Downloaded from http://www.jimmunol.org/content/188/12/5824

Supplementary http://www.jimmunol.org/content/suppl/2012/05/14/jimmunol.110325 Material 3.DC1 http://www.jimmunol.org/ References This article cites 29 articles, 18 of which you can access for free at: http://www.jimmunol.org/content/188/12/5824.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2012 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Cutting Edge: LPS-Induced Emergency Myelopoiesis Depends on TLR4-Expressing Nonhematopoietic Cells ,†,1 ,‡,1 ,† Steffen Boettcher,* Patrick Ziegler,* x Michael A. Schmid,*{ ‖ Hitoshi Takizawa,*,† Nico van Rooijen, Manfred Kopf, Mathias Heikenwalder, and Markus G. Manz*,† Systemic bacterial infection is rapidly recognized as TLRs (6). In addition to their expression and function on an emergency state leading to release into mature hematopoietic cells, such as and den- the circulation and increased myeloid cell production dritic cells, TLRs are expressed on immature hematopoietic within the . However, the mechanisms of stem cells, as well as on myeloid (7), lymphoid (8), and den- sensing infection and subsequent translation into emer- dritic cell (9, 10) progenitor cells, and TLR triggering on gency myelopoiesis have not been defined. In this study, hematopoietic stem and progenitor cells can lead to differ- Downloaded from we demonstrate in vivo in mice that, surprisingly, selec- entiation into mature effector cells (11–14). Importantly, TLR tive TLR4 expression within the hematopoietic com- expression on some nonhematopoietic tissues, such as endo- partment fails to induce LPS-driven emergency myelo- thelial cells and stromal cells, and their involvement in local poiesis. In contrast, TLR4-expressing nonhematopoietic immune responses were also demonstrated recently (15–17). In this study, we addressed the fundamental question of cells are indispensable for LPS-induced, G-CSF–medi- http://www.jimmunol.org/ whether sensing of Gram-negative infection-derived LPS and ated myelopoietic responses. Furthermore, LPS-induced subsequent acute initiation of emergency myelopoiesis depend emergency myelopoiesis is independent of intact IL- on TLR expression within the hematopoietic or nonhema- 1RI signaling and, thus, does not require inflamma- topoietic cellular compartment. some activation. Collectively, our findings reveal a key and nonredundant role for nonhematopoietic compart- Materials and Methods ment pathogen sensing that is subsequently translated Mice and generation of reciprocal chimeras into cytokine release for enhanced, demand-adapted Six- to ten-week-old female wild-type (WT) B6.SJL-PtprcaPep3b/BoyJ + 2/2 + myeloid cell production. The Journal of Immunology, (CD45.1 ) or C57BL/6 TLR4 (CD45.2 ) (18) mice were lethally irra- by guest on September 28, 2021 2 2012, 188: 5824–5828. diated with 13 Gy and transplanted i.v. with 3–5 3 104 sorted Lin c-Kithigh cells of the respective, indicated genotype. All mice were maintained at the Institute for Research in Biomedicine and University Hospital Zurich animal mergency myelopoiesis caused by systemic bacterial in- facility and treated in accordance with guidelines of the Swiss Federal Vet- erinary Office. Experiments were approved by the Dipartimento della Sanita` e fection is characterized by mobilization of Socialita`, Ticino, Switzerland and Veterina¨ramt des Kantons Zurich, Swit- E from the bone marrow (BM), leading to leukocytosis zerland. and neutrophilia. In parallel, BM granulocytic precursors in- Treatment and analysis of mice crease in frequency and accelerate cell cycle progression to re- 2 2 2 2 WT, TLR4 / , IL-1RI / (19), and chimeric mice were injected twice i.p. plenish mature, rapidly consumed neutrophils. These processes with 35 mg LPS (Escherichia coli 0111:B4, Ultrapure; InvivoGen, San Diego, are governed by the concerted action of multiple hematopoietic CA) in a 48-h interval and analyzed 24 h later. Cytospins from peripheral growth factors (1–5). However, it is unclear how increased (PB) were prepared, and May–Grunwald–Giemsa staining was per- formed. For FACS analysis of myelopoietic responses, Gr-1 (RB6-8C5) and hematopoietic growth factor availability and the induction of CD11b (M1/70) Abs were used (both from eBioscience). Myeloerythroid emergency myelopoiesis upon systemic pathogen spread are progenitor FACS analysis and CFU assays were performed, as described (7). 2/2 m regulated. WT and TLR4 mice were injected i.p. with 250 g/kg body weight human G-CSF (Filgrastim; Amgen) six times in a 12-h interval and analyzed Sensing of conserved pathogen-associated molecular pat- with the same method used for the LPS-injected mice. Plasma G-CSF was terns occurs through pattern-recognition receptors, such as analyzed by ELISA, according to the manufacturer’s instructions (R&D

*Institute for Research in Biomedicine, 6500 Bellinzona, Switzerland; †Division of German Academic Exchange Service (D/06/44442 to S.B.), the Swiss National Science , Zurich University Hospital, 8091 Zurich, Switzerland; ‡Hematology Foundation (310000-116637), Oncosuisse (OCS-02019-02-2007), and the Bill and and Oncology, Rheinisch-Westfaelische Technische Hochschule Aachen, 52074 Aachen, Melinda Gates Foundation Grand Challenges in Global Health initiative (to M.G.M.). x Germany; Department of Molecular Cell Biology, VU University Amsterdam, 1081 BT { Address correspondence and reprint requests to Prof. Markus G. Manz, Division of Amsterdam, The Netherlands; Institute of Integrative Biology, Molecular Biomedicine, ‖ Hematology, University Hospital Zurich, Raemistrasse 100, CH-8091 Zurich, Switzer- Swiss Federal Institute of Technology Zurich, 8952 Schlieren, Switzerland; and Insti- land. E-mail address: [email protected] tute for Virology, Technische Universita¨tMu¨nchen/Helmholtz Center Mu¨nchen, 81675 Munich, Germany The online version of this article contains supplemental material. 1S.B. and P.Z. contributed equally to this work. Abbreviations used in this article: BM, bone marrow; PB, peripheral blood; WT, wild- type. Received for publication November 15, 2011. Accepted for publication April 18, 2012. This work was supported in part by the Helmholtz Foundation, the Swiss National Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00 Science Foundation and a starting European Research Council grant (to M.H.), the www.jimmunol.org/cgi/doi/10.4049/jimmunol.1103253 The Journal of Immunology 5825

Systems). For depletion, mice received 400 ml liposomal clodr- and aspirating 10 ml warm PBS into the peritoneal cavity. FcRs were blocked onate i.p. (provided by N.v.R) twice in a 48-h interval (for experimental using purified CD16/32 (93) Ab and stained with Pacific blue-conjugated details see Supplemental Fig. 2E). CD45.1 (A20), FITC-conjugated CD45.2 (104), PE-conjugated F4/80 (BM8), and allophycocyanin-conjugated CD11b (M1/70) Abs (all from Tissue macrophage isolation and analysis eBioscience) to determine donor/recipient chimerism. Lung tissues were embedded in Tissue-Tek OCT compound (Sakura), Single-cell suspensions from BM and spleen were prepared and subjected to frozen in liquid nitrogen, and stored at 280˚C. Five-micron-thick cry- digestion with collagenase D (0.25%; Roche Diagnostics) and DNAse I (20 U/ osections were cut on a Leica CM 1950 Cryostat (Leica) and fixed in acetone. ml; Roche Diagnostics). Hepatic macrophages were isolated, as previously The following Abs were used: CD45.1-Alexa Fluor 488 (A20), CD45.2- reported (20). Peritoneal macrophages were isolated by repeatedly instilling allophycocyanin (104) (both from eBioscience), and CD68 (Serotec) com- Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 1. PB neutrophilia and BM emergency myelopoiesis are exclusively dependent on TLR4-expressing nonhematopoietic cells in vivo but do not require 2 2 intact IL-1R signaling. (A) May–Grunwald–Giemsa staining of cytospin preparations of PB from WT, TLR4 / , and chimeric mice at 72 h after double PBS injection (top panel) or LPS (35 mg from E. coli strain 0111:B4) injection (bottom panel). Original magnification 3250. (B) Percentages of CD11b+Gr-1+ mature 2 2 and immature myeloid cells in PB of PBS- or LPS-injected WT, TLR4 / , and chimeric mice, as described in (A). Mean 6 SEM of two independent experiments, with a total of three PBS- and four LPS-treated mice/group, are shown. (C) Representative FACS profile of CD11b+Gr-1high and CD11b+Gr-1low 2 2 cells in the BM of WT, TLR4 / , and chimeric mice after PBS or LPS injection, as described in (A). Percentages of CD11b+Gr-1high mature (D) and 2 2 CD11b+Gr-1low immature (E) myeloid cells in the BM of PBS- or LPS-treated WT, TLR4 / , and chimeric mice after LPS stimulation. Mean 6 SEM of five independent experiments, with a total of 5 PBS- and 10 LPS-treated mice/group, are shown. (F) Representative FACS profile of CD11b+Gr-1high and 2 2 CD11b+Gr-1low cells in the BM of WT and IL-1RI / mice after PBS or LPS injection, as described in (A). (G) Percentages of CD11b+Gr-1+ mature and 2 2 immature myeloid cells in PB from PBS- or LPS-injected WT and IL-1RI / mice, as described in (A). Mean 6 SEM of three independent experiments, with a total of five PBS- and eight LPS-treated mice/group, are shown. Percentages of CD11b+Gr-1high mature (H) and CD11b+Gr-1low immature (I) myeloid cells in 2 2 the BM of PBS- or LPS-treated WT and IL-1RI / mice after LPS stimulation. Mean 6 SEM of three independent experiments, with a total of five PBS- and eight LPS-treated mice/group, are shown. *p , 0.05, **p , 0.01, ***p , 0.001. 5826 CUTTING EDGE: NONHEMATOPOIETIC CELLS ARE ESSENTIAL IN PATHOGEN SENSING bined with a secondary goat anti-rat–Alexa Fluor 594 Ab (Invitrogen). CD3+ T cells (Supplemental Fig. 1B). Importantly, the vast Immunoselect mounting medium (DAPI; Dianova) was used for embedding majority (.97%) of tissue-resident macrophages in BM, stained cryosections. Images were acquired on a Leica SP5 confocal micro- scope (Leica) and analyzed using Leica LAS AF Lite 2.4 software. Para- spleen, lung, and peritoneal cavity were of donor origin 3 mo formaldehyde-fixed and paraffin-embedded liver sections were analyzed after transplantation (Supplemental Fig. 1C, 1D). However, using F4/80 mAb (BM8). ∼20% of hepatic macrophages were still host derived, con- Equations and statistical analyses sistent with previous findings (20). Chimeric and control mice were challenged with LPS or PBS and analyzed for the first Significance of differences was analyzed with an ungrouped or grouped two- hallmark of emergency myelopoiesis: induction of neutrophilia tailed Student t test or one-sample t test, where appropriate. All statistical and increase in myeloid precursors in the PB. No differences analyses were calculated with GraphPad Software, v5.01 (Prism). 2 2 between nonreconstituted (WT and TLR4 / ) and control- 2 2 2 2 reconstituted (WT/WT and TLR4 / /TLR4 / ) control Results and Discussion 2 2 We generated BM chimeric mice with TLR4 / hematopoiesis mice were observed (data not shown). There was an increase 2 2 on a WT nonhematopoietic background (TLR4 / /WT in mature and immature neutrophils in the PB of WT and 2 2 2 2 mice) and WT hematopoiesis on a TLR4 / nonhemato- TLR4 / /WT mice, as revealed by morphological exami- 2 2 poietic background (WT/TLR4 / mice). At 12 wk after nation of cytospin preparations and by immunophenotypical 2 2 transplantation, no differences in engraftment efficiency or analysis using CD11b and Gr-1 Ags. In contrast, TLR4 / 2 2 lineage reconstitution were observed, and residual host CD45+ and WT/TLR4 / mice failed to mount a respective re-

BM chimerism was ,1% (data not shown, Supplemental Fig. sponse to systemic LPS challenge (Fig. 1A, 1B, Supplemental Downloaded from 1A). Remaining host cells consisted mostly of long-lived Fig. 1E). http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 2. G-CSF is strongly increased upon selective stimulation of TLR4-expressing nonhematopoietic cells and is a major mediator of emergency 2 2 myelopoiesis in vivo. (A) Plasma G-CSF protein levels in PBS- or LPS-treated (35 mg from E. coli strain 0111:B4) WT, TLR4 / , and chimeric mice analyzed 72 h after double injection. Mean 6 SEM of three independent experiments, each with three PBS- and four LPS-treated mice/group, are shown. (B) Plasma G-CSF 2 2 protein levels in PBS- or LPS-treated IL-1RI / mice, treated as described in (A). Mean 6 SEM of two independent experiments, with a total of three PBS- and 2 2 six LPS-treated mice/group, are shown. (C) Representative FACS profile showing myeloid cells (CD11b+Gr-1+) in PB of G-CSF–treated WT mice and TLR4 / mice. The treatment regimen consisted of six injections with 250 mg human G-CSF/kg body weight every 12 h over a period of 3 d. Mice were analyzed 24 h after 2 2 the last injection. (D) Representative FACS profile of CD11b+Gr-1high and CD11b+Gr-1low cells in the BM of WT and TLR4 / mice following G-CSF 2 2 injection, as described in (C). (E) Percentages of CD11b+Gr-1+ mature and immature myeloid cells in PB from PBS- or G-CSF–injected WT and TLR4 / mice, as described in (C). Mean 6 SEM of two independent experiments, with a total of four G-CSF–treated mice, are shown. Percentages of CD11b+Gr-1high mature 2 2 (F) and CD11b+Gr-1low immature (G) myeloid cells in the BM of PBS- or G-CSF–injected WT and TLR4 / mice, as described in (C). Mean 6 SEM of two independent experiments, with a total of four G-CSF–treated mice, are shown. **p , 0.01, ***p , 0.001, ****p , 0.0001. The Journal of Immunology 5827

Next, we studied the second hallmark of emergency mye- To determine whether the massive increase in G-CSF upon lopoiesis: BM myeloid proliferation and differentiation. In LPS stimulation is, as a single cytokine, sufficient to translate 2 2 WT and TLR4 / /WT mice, mature myeloid cells into emergency myelopoiesis, we injected human G-CSF into 2 2 (CD11b+Gr-1high) decreased significantly upon LPS injection, WT and TLR4 / mice. This treatment led to qualitative and whereas the frequency and total numbers of immature pro- quantitative responses that were indistinguishable from those and myelocytes (CD11b+Gr-1low) (21, 22) in- induced by LPS application (Fig. 2C–G, data not shown). creased up to 2.5-fold (Fig. 1C–E, data not shown). In Thus, our data reveal that the primary, indispensable in vivo contrast, no change in cellular composition could be observed sensing site for LPS-induced emergency myelopoiesis is a 2 2 2 2 in TLR4 / or WT/TLR4 / mice, whereas a small, but TLR4-expressing nonhematopoietic cellular compartment that still significant relative, but not absolute, increase in immature translates this signal into massive G-CSF release. We reason 2 2 CD11b+Gr-1low cells was detectable in WT/TLR4 / mice that the few remaining primarily hepatic macrophages do not (Fig. 1E, data not shown). Because -macrophage play a major role in this context, because macrophage-depleted progenitors and CFU-granulocyte increased in WT and mice show a similar myeloid response as do control mice 2 2 2 2 both TLR4 / /WT and WT/TLR4 / mice, signaling (Supplemental Fig. 2D–H). Future studies are needed to through TLR4 in hematopoietic and nonhematopoietic cells clarify the identity and localization of the TLR4-expressing can skew early hematopoiesis toward myeloid differentiation and, likely cytokine-releasing, nonhematopoietic parenchy- (Supplemental Fig. 2A–C). However, nonhematopoietic cell- mal or stromal cell populations. Selective delivery of TLR expressed TLR4 is sufficient, as well as necessary, for induc- mimetics to these cells could then be an intriguing therapeutic Downloaded from tion of full-blown emergency myelopoiesis. possibility to enhance myeloid cell regeneration, supplement- IL-1b is a paradigmatic proinflammatory cytokine driving ing the current need for application of single recombinant the expression of various myelopoiesis-acting cytokines, such cytokines. as G-csf, M-csf, and Gm-csf (23). Thus, initiation of emergency myelopoiesis may involve activation of the inflammasome, leading to IL-1b release that stimulates IL-1RI–expressing cells Acknowledgments http://www.jimmunol.org/ We thank S. Akira (Department of Host Defense, Research Institute for to secrete myelopoiesis-acting growth factors, which, in turn, 2 2 Microbial Diseases, Osaka University) for providing TLR4 / mice. promote emergency myelopoiesis, as suggested recently (24). However, LPS stimulation caused an identical emergency 2 2 myelopoietic response in IL-1RI / mice compared with WT Disclosures mice (Fig. 1F–I, data not shown). Thus, we conclude that The authors have no financial conflicts of interest. IL-1RI signaling may be involved in alternative pathways leading to reactive neutrophilia, such as treatment with the

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Supplemental Figure 1. Overall hematopoietic and macrophage chimerism in different tissues of TLR4-/-;WT and WT;TLR4-/- mice, and PB neutrophilia in WT, TLR4-/-, and chimeric mice.

(A) Representative FACS analysis of CD45.1 and CD45.2 congenic marker expression in the BM of age and sex-matched WT, TLR4-/- and transplanted chimeric TLR4-/-;WT and WT;TLR4-/-mice 12 weeks after transplantation. One representative example from over 30 analyses is shown.

(B) Lineage distribution of remaining host-derived cells in chimeric animals. Mean

± SEM of three independent experiments with a total of nine transplanted animals is shown.

(C) Confocal microscopy analysis showing chimerism in chimeric mice based on expression of CD68. Representative results from one analysis out of two are shown.

(D) Flow cytometric analysis of macrophage chimerism in BM, spleen, peritoneal cavity, and liver of chimeric miceat 12 weeks after transplantation. One representative analysis out of three experiments is shown.

(E)Representative FACS profile showing myeloid cells (CD11b+Gr-1+) in peripheral blood from PBS or LPS-injected WT, TLR4-/-, and chimeric mice.

Non-hematopoietic cells are essential for pathogen sensing 1 Boettcher et al. Supplemental Online Materials

Supplemental Figure 2.GMP frequenciesand absolute CFU numbers in the

BM of WT, TLR4-/- and chimeric mice, and emergency myelopoiesis in macrophage-depleted WT mice.

(A)Representative FACS profile showing -erythrocyte progenitors

(MEPs, CD34-FcgRlow), common myeloid progenitors (CMPs, CD34+FcgRlow), and granulocyte-macrophage progenitors (GMPs, CD34+FcgRhigh) in the BM of

PBS or LPS-treated control and chimeric mice. Cells were pre-gated on Lin-c-

KithighSca-1- population. One representative result out of two independent experiments is shown.

(B)GMP frequencies in the BM of PBS-treated (white circles) WT, TLR4-/-, and chimeric miceor after LPS stimulation (grey circles). Results of two independent experiments with a total of three PBS and four to five LPS-treated mice per group are shown.

(C) CFU numbers per hind leg (femur + tibia) of PBS-treated WT, TLR4-/-, and chimeric mice or after LPS stimulation as indicated. Results of two independent experiments with a total of two to three mice per group are shown.

(D) Experimental outline depicting protocol for macrophage depletion and subsequent LPS stimulation. Briefly, WT mice were i.p. injected twice with 400μl liposomal clodronate (LipClo) or PBS-containing liposomes (empty Lip) in a 48 hour interval. On the day of the second LipClo or empty Lip injection, mice received also 35μg LPS followed by a second LPS injection 48 hours later.

Finally, mice were analyzed for myeloid responses on day 3.

Non-hematopoietic cells are essential for pathogen sensing 2 Boettcher et al. Supplemental Online Materials

(E) FACS analysis showing macrophage compartments (CD11bintF4/80high) in

BM, spleen and liver two days after a single i.p. injection of liposomal clodronate

(LipClo) or control liposomes containing only PBS (empty Lip). One representative analysis out of two independent experiments is shown.

(F) Immunohistochemical analysis showing macrophages (F4/80+) in liver sections from LipClo-treated and control mice receiving empty Lip. One representative analysis out of two experiments is shown. Original magnification,

250x.

(G) Emergency myelopoietic responses as determined by percentage of PB myeloid cells (CD11b+Gr-1+) in control, empty Lip-, and LipClo-treated mice either receiving LPS (grey circles) or PBS (white circles).Results of two independent experiments are shown.

(H) Representative FACS analysis showing percentages of myeloid cells

(CD11b+Gr-1+) in PB of control, empty Lip-, and LipClo-treated mice either receiving LPSor PBS. One representative example out of two independent experiments with 3-5 mice per group is shown.

Non-hematopoietic cells are essential for pathogen sensing 3 Supplemental Figure 1, Boettcher et al.

BM A B BM WT TLR4-/- TLR4-/- WT WT TLR4-/-

5 100 10 0 97.3 98.7 0.6 CD3 B220 104 80 not specified 97.3 0 0.5 97.6 103 60 CD45.2

102 40 0 of remaining 0102 103 104 105 0102 103 104 105 0102 103 104 105 0102 103 104 105 20

CD45.1 Lineage distribution host-derived cells (%) 0

C Lung

TLR4-/- WT

CD45.1 50μm CD45.2 50μm CD68 50μm CD68 CD45.1 50μm CD68 CD45.2 50μm

WT TLR4-/-

CD45.1 50μm CD45.2 50μm CD68 50μm CD68 CD45.1 50μm CD68 CD45.2 50μm

D TLR4-/- WT WT TLR4-/-

5 5 5 5 10 1.0 10 10 0.94 10 2.2 104 104 104 104 96.8

3 BM 103 103 10 103

2 2 10 102 96.2 10 102 2 0 0 0 0

5 5 5 5 10 0.9 10 10 0.5 10 1.3 104 104 104 104 98.3

3 3 Spleen 10 103 10 103

2 2 10 102 98.4 10 102 1.7 0 0 0 0 F4/80 F4/80 CD45.1 CD45.1 5 5 5 5 10 25.7 10 10 27.9 10 0.1 104 104 104 104 99.9

Peritoneal 3 3 10 103 10 103 Macrophages 2 2 10 2 10 2 10 10 0.09 0 99.8 0 0 0 105 9.8 105 105 7.5 105 22 80.2 4 4 10 104 10 104

103 103 Liver 103 103

0 0 0 77.3 0 19.6

0102 103 104 105 0102 103 104 105 0102 103 104 105 0102 103 104 105 CD11b CD45.2 CD11b CD45.2

E PB WT TLR4-/- TLR4-/- WT WT TLR4-/- 105 15.7 10.2 14.7 17.5

104 PBS 103

102

0

5 Gr-1 10 45 10.3 49.1 13.2

104 LPS 103

102

0

0102 103 104 105 0102 103 104 105 0102 103 104 105 0102 103 104 105 CD11b