Selective Expansion of the Monocytic Lineage Directed by Bacterial Infection Natalya V. Serbina, Tobias M. Hohl, Mathew Cherny and Eric G. Pamer This information is current as of September 25, 2021. J Immunol 2009; 183:1900-1910; Prepublished online 13 July 2009; doi: 10.4049/jimmunol.0900612 http://www.jimmunol.org/content/183/3/1900 Downloaded from

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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 © 2009 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Selective Expansion of the Monocytic Lineage Directed by Bacterial Infection1

Natalya V. Serbina,2,3 Tobias M. Hohl,2 Mathew Cherny, and Eric G. Pamer

CCR2-mediated recruitment of Ly6Chigh is essential for defense against a range of microbial pathogens. Although our understanding of trafficking to inflammatory sites is increasing, how innate immune inflammation influences monocyte development and maturation during microbial infection remains undefined. Herein, we demonstrate that infection with the intracellular bacterial pathogen Listeria monocytogenes specifically and selectively promotes monopoiesis. Systemic infection with virulent L. monocytogenes induces marked proliferation of monocyte precursors and results in depletion of myeloid progenitors. Proliferation of monocyte precursors correlates with the intensity of systemic infection and is unaffected by the density of monocytes in the bone marrow. Although MyD88/Trif-mediated signaling is not required for early emigration of the

mature monocyte population from the bone marrow, replenishment of monocyte populations depends on MyD88/Trif. Our studies Downloaded from demonstrate that TLR-mediated signals play an essential role in the maintenance of monocyte homeostasis during systemic bacterial infection. The Journal of Immunology, 2009, 183: 1900–1910.

onocytes arise from myeloid progenitors in the bone via CCR2, requires cytoplasmic invasion by bacteria and does not marrow and are released into the peripheral circulation occur when heat-killed or noninvasive bacteria are administered to (1). Bloodstream monocytes are heterogeneous and mice (5, 6). M ϩ http://www.jimmunol.org/ distinct subsets can be defined by expression of CD11b, Ly6C, 7/4, During systemic infection with L. monocytogenes, CCR2 F4/80, and several chemokine receptors (2). The CCR2 chemokine monocytes are recruited to the spleen and differentiate into TNF- receptor is expressed on a subset of circulating monocytes that and inducible NO synthase (iNOS)4-producing dendritic cells (7) traffic to sites of inflammation. CCR2ϩ monocyte trafficking is in a process that depends on dendritic cells and NK cells (8). TNF- induced by some infections and can be essential for protection and iNOS-producing dendritic cells are the major source of TNF against intracellular bacterial pathogens, including Listeria mono- and iNOS during L. monocytogenes infection and failure to recruit cytogenes (3). these cells leads to unrestricted bacterial replication and impaired L. monocytogenes is a Gram-positive facultative intracellular host survival. Recent studies demonstrated recruitment of TNF- bacterium that infects a wide range of invertebrate and vertebrate and iNOS-producing monocytes to gut-associated tissues follow- by guest on September 25, 2021 hosts. Originally identified as a causative agent of a lethal disease ing infection with Salmonella typhimurium or Toxoplasma gondii in rabbits, it was noted to induce marked monocytosis in infected (9, 10), and CCR2-dependent monocyte recruitment is critical for animals; hence its name. Murine infection with L. monocytogenes control of T. gondii infection (9). Thus, CCR2ϩ monocytes may has been used extensively as a model for studying immune re- play a more general role in innate immune responses to microbial sponses to intracellular bacterial pathogens. Innate immunity is infections. rapidly triggered following infection and is crucial for initial con- Following infection with L. monocytogenes, CCR2-mediated trol of bacterial replication. Following invasion of mammalian signals promote monocyte exit from bone marrow to the pe- cells, L. monocytogenes initiates escape from the vacuole by se- ripheral circulation. Emigration of mature monocytes from the creting listeriolysin O, an essential virulence factor (4). Escape of bone marrow is more rapid than monocyte replenishment from L. monocytogenes to the cytosol is required for the induction of progenitors, resulting, during the first 24–48 h of infection, in proinflammatory mediators and establishment of protective immu- markedly diminished monocyte numbers in the bone marrow nity. Secretion of MCP-1 and MCP-3, two chemokines that signal (11). How monocyte populations are replenished during the course of infection is unclear. During normal hematopoiesis, monocytes arise from a recently described subset of lineage- Infectious Disease Service, Department of Medicine, Memorial Sloan-Kettering Can- negative bone marrow progenitors referred to as cer Center, Immunology Program, Sloan-Kettering Institute, New York, NY 10021 and progenitors (MDPs), which express c-kit (re- Received for publication February 25, 2009. Accepted for publication May 27, 2009. ceptor for stem cell factor) and chemokine receptor CX3CR1 The costs of publication of this article were defrayed in part by the payment of page (12). This subset also expresses CD115 (M-CSF receptor) and charges. This article must therefore be hereby marked advertisement in accordance can express receptor tyrosine kinase Flt3 (Flk2, CD135) (13). with 18 U.S.C. Section 1734 solely to indicate this fact. The fate of MDPs and their progeny following infection has not 1 The authors’ research is supported by the National Institutes of Health (Grants R37 been examined. AI39031 and P01 CA023766-30 to E.G.P; K12 CA120121 to N.V.S.; and K08 AI071998 to T.M.H.) and Irvington Institute for Immunological Research (to N.V.S.). In this study, we report that monocytes are continuously re- N.V.S. conceived, designed, and performed research, analyzed data, and wrote the cruited to the spleens during the course of L. monocytogenes paper; T.M.H. generated CCR2 reporter mice; M.C. performed research; and E.G.P. designed research and wrote the paper. 4 2 Abbreviations used in this paper: iNOS, inducible NO synthase; MDP, macrophage N.V.S. and T.M.H. contributed equally. and dendritic cell progenitor; BAC, bacterial artificial chromosome; miRNA, mi- 3 Address correspondence and reprint requests Dr. Natalya V. Serbina, Infectious croRNA; ICE, IL-1␤-converting enzyme. Diseases Service, Memorial Sloan-Kettering Cancer Center, Sloan Kettering Institute, 1275 York Avenue, New York, NY 10021. E-mail address: [email protected] Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00 www.jimmunol.org/cgi/doi/10.4049/jimmunol.0900612 The Journal of Immunology 1901 infection. Monocyte-mediated immunity is comprised of two Flow cytometry distinct phases. In the first phase, significant numbers of pre- Spleens were harvested at indicated times postinfection, dissociated, and existing monocytes are recruited from the bone marrow to the digested with 0.3% collagenase type 4 (Worthington Biochemical). Bone periphery in response to infection. During the second phase, marrow cells were harvested from mouse femurs. Erythrocytes were lysed infection of mice results in rapid, robust but selective expansion with NH4Cl-Tris solution and cells were stained for cell surface and in- of monocyte precursors in the bone marrow, leading to contin- tracellular markers. The following Abs were purchased from BD Pharm- ingen: anti-CD11b (M1/70), anti-CD11c (HL3), anti-B7-1 (16-10A1), anti- uous replenishment of monocytes in the periphery. Monocyte B7-2 (GL1), anti-I-A/I-E (M5/114.15.2), anti-Ly6G (1A8), anti-Ly6C expansion is not due to general expansion of the myeloid com- (AL-21), anti-c-kit (104D2), anti-Sca-1 (D7), and anti-Flk2 (A2F10.1). partment because the number of bone marrow is Anti-CD115 (AFS98) was purchased from eBioscience. Anti-F40/80 concurrently reduced. Infection-induced monopoiesis requires (A3-1) and anti-monocyte/ (7/4) were purchased from Serotec. The lineage Ab mixture included the following Abs (BD Pharmingen): MyD88-dependent and IL-1/IL-18- independent signaling. anti-LyC and Ly6G (RB6-8C5), anti-CD11b (M1/70), anti-CD3e (145- These results suggest that inflammation induced by L. monocy- 2C11), anti-CD45/B220 (RA3-6B2), anti-erythroid cells (TER-119), and togenes infection redirects the development of individual he- anti-NK 1.1 (PK136). For morphological examination, cell subsets were matopoietic lineages in bone marrow by specifically promoting obtained by fluorescence-activated sorting with the purity of Ͼ98%. Cells monopoiesis to sustain delivery of monocytes to peripheral sites were spun onto glass slides and stained using Diff-Quik Satin Set (Dade Behring). of infection. In vitro cell culture Sca-1ϩLy6CϪ, Sca-1ϩLy6CϩCD11blow, and Sca-1ϩLy6CϩCD11bhigh Materials and Methods cells were sorted from bone marrows of wild-type mice infected for 3 Downloaded from Mice and infections days. Sorted cells were plated at 105 cells/well in 96-well, flat-bottom Ϫ Ϫ plates in DMEM (Life Technologies) supplemented with 10% FCS, C57BL/6 and CCR2 / mice were bred at Memorial Sloan-Kettering Re- L-glutamine, HEPES, 2-ME, antibiotics (penicillin, streptomycin, gen- search Animal Resource Center. Triflps2/lps2 mice were a gift from Dr. B. tamicin) and recombinant growth factors (R&D Systems), and cells Beutler (The Scripps Research Institute, University of California, San Di- Ϫ Ϫ were cultured for 72 h. The resultant cultures were harvested and an- ego, CA) and bred in-house to MyD88 / mice obtained from Dr. S. Akira alyzed by flow cytometry. (University of Osaka, Osaka, Japan). All mice were backcrossed at least 10

Ϫ Ϫ http://www.jimmunol.org/ generations onto the C57BL/6 background. ICE / on the NOD/ShiLt BrdU incorporation assay background and BALB/cJ mice were purchased from The Jackson Labo- ratory. Mice were infected i.v. with 3000 L. monocytogenes strain 10403S In vivo BrdU labeling and staining was conducted using BrdU Flow Kits (provided by Dr. D. Portnoy, University of California, Berkeley, CA). For from BD Pharmingen. Briefly, mice were administered 1 mg of BrdU so- infection of MyD88Ϫ/ϪTriflps2/lps2 mice and corresponding controls, a lution via the i.p. route and femurs were collected 1–2 h later. Single-cell lower dose of 1000 L. monocytogenes was used. When indicated, ampi- bone marrow suspensions were prepared and processed for cell surface Ag cillin (Sigma-Aldrich) was given to mice at 2 mg/ml in drinking water. All staining followed by staining for incorporated BrdU according to the man- animal experiments were approved by the Institutional Animal Care and ufacturer’s protocol. Use Committee. Results

Generation of CCR2 reporter mice Dynamics of bone marrow monocyte populations following by guest on September 25, 2021 infection with L. monocytogenes CCR2 reporter mice were generated by bacterial artificial chromosome (BAC)-mediated transgenesis using the recombineering strategy pioneered Bone marrow monocytes are characterized by high levels of Ly6C by Heintz and colleagues (14). Briefly, the endogenous CCR2 locus was and intermediate levels of CD11b expression. Under homeostatic identified on BAC clone RP23-182D4 (ϳ250 kb in size; obtained from conditions, Ly6Chigh monocytes represent 5–8% of the total nu- CHORI) derived from the distal end of chromosome 9 by PCR screening cleated bone marrow cells and are ϳ5-fold less frequent than of candidate BACs mapped to this region. A 1.7-kb fragment that con- ϩ tained 1.0 kb upstream and 0.7 kb downstream of the CCR2 gene start Ly6G (35–50% of bone marrow cells; data not high codon was amplified and modified between the first and second codon of shown). In uninfected mice, Ly6C monocytes express 7/4, CCR2 through insertion of nucleotides that encoded aa 2–238 of enhanced M-CSF receptor CD115, and do not express the GFP. The GFP coding sequence was followed by a stop codon and the marker Ly6G (Fig. 1A). Additionally, Ly6Chigh bone marrow ensuing nucleotide at position 4 of the endogenous CCR2 locus was de- leted. The resulting 2.4-kb fragment was cloned into the shuttle vector monocytes are immature and do not express MHC class II, co- pLD53SC.AB obtained from Dr. D. Littman (New York University, New stimulatory molecules, or CD11c (Fig. 1A). Bone marrow mono- York, NY) (14). cytes express the chemokine receptor CCR2 and require CCR2- To modify BAC clone RP23-182D4 for transgenesis, the shuttle vec- mediated signals for bone marrow egress during infection (11). To tor containing the 2.4-kb CCR2.GFP insert was integrated into the BAC facilitate detection of CCR2-expressing monocytes, we generated by homologous recombination and cointegrates were identified by chloramphenicol and ampicillin selection. Resolution of cointegrates transgenic mice in which GFP expression is controlled by the high through a second homologous recombination event was achieved by CCR2 promoter. In these CCR2 reporter mice, Ly6C mono- negative selection on sucrose, resulting in the complete excision of the cytes are uniformly labeled with GFP, confirming that they repre- shuttle vector backbone that includes the SacB gene (for details, see sent a homogeneous population. Sorted Ly6Chigh monocytes are of Ref. 14). The resulting modified BAC encoding GFP under control of CCR2 uniform morphology (Fig. 1A). the endogenous promoter and regulatory elements was analyzed high by Southern blotting to verify GFP integration at the expected site, L. monocytogenes infection induces recruitment of Ly6C sequenced over the CCR2 gene locus, purified by centrifugation through monocytes from the bone marrow to sites of infection (7). To a cesium chloride gradient, and injected into fertilized C57BL/6J oo- determine the extent of infection-induced bone marrow monocyte cytes. Four potential founder animals were identified among 39 off- depletion, bone marrow cellularity was measured at various times spring screened by PCR and flow cytometric analysis of peripheral samples. following systemic L. monocytogenes infection. Total numbers of One candidate, designated CCR2.GFP.8, transmitted the transgene to bone marrow cells in infected mice declined between days 1 and 4 one-half of the progeny and was the founder of the CCR2 reporter after infection (Fig. 1B) and the percentage of Ly6Chigh monocytes colony. CCR2 reporter mice were maintained under specific pathogen- declined for the first 2 days following infection. Three and 4 days free conditions on standard laboratory chow. For specific experiments, Ϫ/Ϫ lps2/lps2 after infection, however, the frequency and total number of the CCR2 reporter mice were bred to MyD88 Trif mice on the high C57BL/6J background. CCR2 reporter MyD88Ϫ/ϪTriflps2/lps2 animals Ly6C monocytes increased (Fig. 1, C and D). In contrast to the were maintained on Sulfatrim feed before use in experiments. increasing number of bone marrow monocytes, the total number of 1902 MONOPOIESIS DURING MICROBIAL INFECTION Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021 FIGURE 1. Kinetics of bone marrow monocytes and granulocytes following infection. A, Left panel, bone marrow cells were harvested from uninfected C57BL/6 mice. Expression of CD11b and Ly6C on the bone marrow leukocytes was analyzed by flow cytometry. Histograms: cells were gated on Ly6C and CD11b (gate R1) and expression of various markers was analyzed (black lines, indicated Abs; shaded areas, control IgGs). Right panel, Cytospins of cells sorted from gate R1 were stained with Giemsa for morphological assessment. Original magnification, ϫ600. Bone marrow cells (B–E) and (F–H) were harvested from C57BL/6 mice infected with 3,000 live L. monocytogenes at indicated times and expression of Ly6C, CD11b, and Ly6G was analyzed. B, Total numbers of leukocytes in the femur. Percentages (C) and total numbers (D) of Ly6ChighCD11bϩ cells in the femurs. E, Total numbers of Ly6CintCD11bϩ cells in the femurs. Percentages (F) and total numbers (G) of Ly6ChighCD11bϩ cells in the spleens. H, Total numbers of Ly6Gϩ cells in the spleens. For all graphs, each time point represents three mice (error bars, SD). This experiment was repeated twice with similar results. Value of p .Ͻ0.001. The unpaired Student t test was used to compare groups ,ءءء ;0.01–0.001 ,ءء ;0.05–0.01 ,ء ;(compared with day 0: Ͼ0.05, not significant (ns Statistical analysis was performed with Prism (GraphPad Software). granulocytes in bone marrow remained low for the first 4 days of cells in the bone marrow of L. monocytogenes-infected mice, how- systemic infection (Fig. 1E), suggesting that monopoiesis but not ever, remained stable for the first 2 days after infection and then was induced by L. monocytogenes infection. Be- rapidly declined (Fig. 2A). We next examined the frequencies of cause monocytes are continuously emigrating from bone marrow lineage-negative bone marrow progenitors that can give rise to to peripheral sites during infection, the static measure of bone mar- monocytes. In the bone marrow, multipotent progenitors (c-kitϩ row cellularity likely underestimates the number of newly gener- Sca-1ϩCD127ϪFc␥RϪ) give rise to common myeloid progenitors ated monocytes. Indeed, marked monocyte recruitment to the (c-kitϩ Sca-1ϪCD127ϪFc␥RϪ) which further differentiate into spleen was observed between days 2 and 4 after infection (Fig. granulocyte/macrophage progenitors (c-kitϩSca-1ϪCD127ϪFc␥Rϩ) 1F), leading to dramatic increases in the total number of splenic (15). A recently identified subset of progenitor cells expressing monocytes (Fig. 1G). In contrast, peak granulocyte recruitment to c-kit, CD115. and CX3CR1 (MDPs) (12, 13) can differentiate into the spleen was detected on the second day of infection and sub- monocytes, , and dendritic cells but not granulocytes sequently decreased (Fig. 1H). and is derived from granulocyte/macrophage progenitors. In the uninfected bone marrow, c-kitϩCD115ϩ cells represented ϳ25% Depletion of lineage-negative monocyte progenitors during of the lineage-negative population (Fig. 2B). These cells expressed infection Fc␥R and did not express CD127 (data not shown) and thus phe- We reasoned that the increase in the numbers of bone marrow notypically resembled MDPs (16, 17). Surprisingly, expression of Ly6Chigh monocytes following infection might result from an ex- CD115 in the LinϪc-kitϩ populations diminished dramatically be- pansion of lineage-negative progenitors committed to the mono- tween days 1 and 2 following infection and CD115ϩ cells reap- cyte/macrophage lineage. The total number of lineage- negative peared only at day 4 (Fig. 2B). Overall, we observed a drastic The Journal of Immunology 1903

FIGURE 2. Infection induces de- pletion of early progenitors in femurs. C57BL/6 mice were infected with L. monocytogenes and femurs were re- moved at the indicated times. A, Ex- pression of lineage markers was ana- lyzed by flow cytometry and total numbers of lineage-negative cells were calculated. Each time point rep- resents three mice (error bars, SD) and the experiment was repeated three times. B, Cells were stained for lineage markers and gated on lin- eage-negative populations. Expres- sion of c-kit and CD115 in LinϪ gate was analyzed. C, Total numbers of LinϪc-kitϩCD115ϩ in a Downloaded from femur. Each time point represents three mice (error bars, SD) and the experiment was repeated two times. Value of p compared with day 0: –0.01 ,ء ;(Ͼ0.05, not significant (ns ,ءءء and ;0.01–0.001 ,ءء ;0.05 Ͻ0.001. The unpaired Student t test http://www.jimmunol.org/ was used to compare groups. Statis- tical analysis was performed with Prism (GraphPad Software). by guest on September 25, 2021 decrease in the number of c-kitϩCD115ϩ monocyte progenitors amined the presence of LinϪc-kitϩFlk2ϩ cells in the bone marrow following infection (Fig. 2C). Because monocytic progenitors also at various times after infection. We found that the number of these express tyrosine kinase Flk2 (Flt3) (12, 18), we additionally ex- cells was also significantly reduced between days 1 and 4 (data not

FIGURE 3. Monocyte precursors proliferate extensively following infection. Naive mice or mice inoculated with L. monocytogenes were administered BrdU at the indicated days and femurs were harvested 1–2 h later. Cells were stained for BrdU, Ly6C (A and B) and CD11b (C). B, Cells were gated on Ly6C and BrdU incorporation within the gated population was analyzed in naive mice and mice infected for 3 days. C, Total numbers of BrdUϩLy6Chigh CD11bϩ, BrdUϩLy6ChighCD11bϪ, and BrdUϩLy6Cint cells in the femur. Each time point represents three mice (error bars, SD) and the experiment was The unpaired Student t test was used .0.01–0.001 ,ءء and 0.05–0.01 ,ء ;(repeated two times. Value of p compared with day 0: Ͼ0.05, not significant (ns to compare groups. Statistical analysis was performed with Prism (GraphPad Software). 1904 MONOPOIESIS DURING MICROBIAL INFECTION

FIGURE 4. Characterization of bone marrow monocyte precursors. Femurs were harvested from naive C57BL/6 mice or mice infected with L. monocytogenes. A and B, Expres- sion of the indicated surface markers on bone marrow leukocytes. Shown are representative dot plots. Three mice were analyzed for each time point and the experiment was re- peated four times. C, Cells harvested 2 and 3 days after infection were gated on the Sca-1highLy6Chigh popu- lation and CD11b expression in the

gate was analyzed. D, Bone marrow Downloaded from cells were harvested 3 days following infection and expression of Sca-1, Ly6C, CD115, CD11b, and CCR2 was analyzed by flow cytometry. Cells were gated on the Sca-1high Ly6Chigh population (R1) and expres- sion of CD115, CD11b, and CCR2 in http://www.jimmunol.org/ the R1 gate was analyzed. E, Bone marrow cells were harvested on day 2 following infection and stained for various surface markers. Cells were sorted from the gates indicated above the panels and cytospins were pre- pared. The experiment was repeated two times. by guest on September 25, 2021

shown). Thus, inflammation did not lead to a rapid replenishment significantly between days 1 and 3 after infection (Fig. 3C). Al- of the immature progenitor pool. though most BrdUϩ cells on day 2 of infection were Ly6Chigh CD11bϪ, by day 3 the BrdUϩ population was comprised of both Proliferation of monocyte populations in the bone marrow CD11bϪ and CD11bϩ populations (Fig. 3C). At the same time, the following infection number of proliferating Ly6Cint cells (granulocytes) decreased Under homeostatic conditions, mature monocytes are the nonpro- (Fig. 3C). Thus, L. monocytogenes infection specifically promotes liferating progeny of dividing and . We expansion of monocytes while suppressing proliferation of other next examined whether Ly6Chigh cells were actively dividing dur- cell lineages. ing the course of infection. To assess cellular proliferation, BrdU was administered to mice at distinct times following infection and Characterization of monocytes in bone marrow following bone marrow cells were collected 1–2 h later. The short duration infection of BrdU pulse allowed us to estimate the proportion of cells en- Although bacterial infection decreases the frequency of lineage- tering and progressing through the S phase of the cell cycle at the negative monocyte progenitors in bone marrow, the effect on time of bone marrow harvest. In uninfected mice and in mice in- the frequency of later stage monocyte progenitors and mono- fected for 1 day, the majority of DNA-synthesizing cells were cyte maturation is unknown. We reasoned that newly replen- Ly6CϪ and Ly6Chigh cells represented only a small fraction of ished monocytes may retain expression of some markers of lin- these cells (Fig. 3A). By day 2 of infection, the proportion of eage-negative monocyte precursors. Because Sca-1 (Ly6 A/E) Ly6Chigh cells increased within the BrdUϩ pool and a concomitant and c-kit have both been useful markers to distinguish bone decrease was observed in proliferating Ly6CϪ cells. As infection marrow stem cells and progenitors, we chose to examine the progressed, the incorporation of BrdU was predominantly in expression of these Ags on bone marrow populations. We there- Ly6Chigh cells, and these cells accounted for ϳ70% of the total fore investigated monocyte differentiation by characterizing co- cells undergoing DNA synthesis (Fig. 3A and data not shown). expression of lineage markers and either c-kit or Sca-1 on bone Within the Ly6Chigh population, a significantly higher fraction of marrow cells 1, 2, and 3 days following systemic L. monocy- cells was driven to divide by day 3 following infection (Fig. 3B). togenes infection. Although expression of lineage markers was The number of Ly6Chigh cells that incorporated BrdU increased associated with down-modulation of c-kit (data not shown), we The Journal of Immunology 1905

FIGURE 5. Differentiation poten- tial of Sca-1high progenitors. Mice were infected with L. monocytogenes and femurs were collected 3 days later. Indicated populations were puri- fied by sorting and cultured for 3 days in the presence of growth factors or

medium alone. At the end of culture, Downloaded from total cell numbers were calculated (A) and forward scatter parameters (B) and expression of the indicated cell surface markers (C) were analyzed by flow cytometry. Experiment was re- peated once with similar results. http://www.jimmunol.org/ by guest on September 25, 2021

detected a dramatic increase in the frequency of Sca-1highLinϩ population also contained cells with ring-shaped nuclei with a cells in the bone marrow of mice 2 and 3 days after infection karyoplasmic ring wider than the diameter of its center, a mor- (Fig. 4A). We next characterized the Sca-1high population in phology that is characteristic of the mononuclear lineage (19) greater detail. In naive mice and in mice 1 day following in- (Fig. 4E). Sca-1highLy6ChighCD11bϩ cells were larger in size fection, very few Sca-1high cells were present in the bone mar- with less basophilic cytoplasm, more abundant vacuoles. and row (Fig. 4B and data not shown). The percentage of Sca-1high some pseudopods. The nucleus:cytoplasm ratio in this cell pop- cells increased 2 and 3 days after infection and most of these ulation was ϳ1orϾ1 and nuclei had a horseshoe or kidney cells expressed high levels of Ly6C (Fig. 4B). Only a fraction shape (Fig. 4E). Thus, Sca-1highLy6ChighCD11bϩ cells repre- of Sca-1highLy6Chigh cells, however, expressed CD11b at days sent populations of immature monocytes while Sca-1highLy6Chigh 2 and 3 after infection (Fig. 4C). To determine whether Sca- CD11bϪ cells are monoblasts. 1highLy6Chigh cells represent inflammatory monocyte progeni- In vitro differentiation of Sca-1highLy6ChighCD11bϪ/low and tors, expression of CCR2, CD115, and CD11b by this popula- high high ϩ tion was further examined. At day 3 after infection, the majority Sca-1 Ly6C CD11b cells of Sca-1highLy6Chigh cells expressed CCR2 and CD115 and het- The up-regulation of Ly6C and CD11b expression by Sca-1high erogeneous levels of CD11b (Fig. 4D). To confirm that Sca- cells could result from sequential differentiation of Ly6CϪ 1high cells represent newly replenishing monocytes, prolifera- CD11bϪ cells toward Ly6ChighCD11bϪ and then toward Ly6Chigh tion of these cells was also examined. We observed significant CD11bϩ cells. Alternatively, these cells may represent distinct dif- BrdU incorporation by Sca-1high cells 3 days following infec- ferentiation branches that are not linearly related. To determine tion and the percentage of Ly6ChighBrdUϩ cells correlated the potential of Sca-1highLy6CϪCD11bϪ and Sca-1highLy6Chigh closely with the percentage of Sca-1highBrdUϩ cells (data not CD11bϪ cells to differentiate into CD11b-expressing monocytes/ shown). macrophages, we sorted Sca-1highLy6CϪCD11bϪ, Sca-1highLy6Chigh Sca-1highLy6Chigh CD11bϪ/low and Sca-1highLy6ChighCD11bϩ CD11bϪ, and Sca-1highLy6Chigh CD11bϩ cells from bone marrow of cells were sorted from bone marrow 2 days after infection for mice infected for 2 days with L. monocytogenes. Sorted cells were morphological analysis. Sca-1highLy6ChighCD11bϪ cells were cultured in vitro for 24–48 h in the presence of M-CSF or GM-CSF. small round cells with a small rim of basophilic cytoplasm, few Culture in the presence of either growth factor led to survival of all cytoplasmic vesicles, and a Ͼ1 ratio of nucleus:cytoplasm. This sorted cell populations with the best recovery observed for the 1906 MONOPOIESIS DURING MICROBIAL INFECTION Downloaded from http://www.jimmunol.org/

FIGURE 6. Monocyte reconstitution requires ongoing inflammation. CCR2Ϫ/Ϫ mice were infected with L. monocytogenes (LM) and ampicillin (amp) was administered to one group of mice 24 h after infection. Bone marrow cells were harvested from uninfected mice, mice infected for 3 days, and infected mice that received antibiotics and the numbers of total cells were counted (A). B, Expression of Sca-1, Ly6C, CD11b, CD115, and F4/80 was analyzed by flow cytometry. by guest on September 25, 2021 Right panels, Cells were gated on the Ly6ChighCD11bϩ population (R1) and expression of CD115 and F4/80 in the R1 gate was analyzed. C, The numbers of Ly6ChighCD11bϩ bone marrow cells were calculated. Each bar represents the average of three mice and error bars show SDs. D, Naive and Listeria-infected mice were administered BrdU and femurs were harvested 1.5 h later. Cells were stained for BrdU, Ly6C, and CD11b. Upper panels, BrdU and Ly6C staining in the bone marrow leukocytes. Lower panels, Ly6C and CD11b expression in cells gated on BrdU. E, Total numbers of BrdUϩ and BrdUϩLy6Chigh cells in the femurs. Each bar represents three mice (error bars, SD). The unpaired Student t test was used to compare groups. Statistical analysis was performed with Prism (GraphPad Software).

Sca-1highLy6CϩCD11bϪ population (Fig. 5A). Cultures of Sca-1high bone marrow of infected CCR2-deficient mice because monocyte Ly6CϪ cells were characterized by small forward scatter properties emigration to the periphery is impaired in these mice (11, 20). This characteristic of lymphocytes while cells derived from Sca-1high enabled us to measure monocyte expansion in bone marrow with- Ly6ChighCD11bϪ and Sca-1highLy6Chigh CD11bϩ populations were out the potential confounding effect of unmeasured monocyte em- significantly larger (Fig. 5B). Despite enhanced survival of igration during the course of infection. Sca-1highLy6CϪ cells in the presence of M-CSF and GM-CSF, very To directly address whether progressive infection is required for few Ly6ChighCD11bϩF4/80ϩ cells could be recovered from resultant monopoiesis, infected mice were treated with oral ampicillin 24 h cultures (Ͻ5%), suggesting that Sca-1highLy6CϪ cells did not have in after initiation of infection. Treatment with ampicillin leads to bac- vitro macrophage differentiation potential (Fig. 5C). In contrast, cul- terial clearance and reduction of infection-associated inflammation tures of Sca-1highLy6Chigh CD11bϪ and Sca-1highLy6Chigh CD11bϩ within 12 h of administration (21). Antibiotic administration did cells yielded a similar frequency of CD11bϩF4/80ϩ cells, suggesting not interfere with the initial phase of cellular emigration from the that Sca-1highLy6ChighCD11bϪ cells can differentiate toward CD11b- bone marrow since comparable reductions in total cell numbers expressing macrophages. Thus, the bone marrow population of were observed in antibiotic-treated and untreated mice (Fig. 6A). Ϫ Sca-1highLy6ChighCD11b cells represents progenitors of Three days after infection of CCR2Ϫ/Ϫ mice, Sca-1highLy6Chigh monocytes. and Ly6ChighCD11bϩ/Ϫ cells represented ϳ50% of the bone mar- row population (Fig. 6B) and their numbers were significantly in- Monocyte expansion requires ongoing inflammation creased compared with naive controls (Fig. 6C and data not It is possible that monocyte proliferation and differentiation require shown). Newly generated Ly6ChighCD11bϩ/Ϫ cells expressed continuous inflammation or progressing infection. Alternatively, CD115 and a significant proportion of these cells expressed F4/80, bone marrow monopoiesis may be stimulated during early stages indicating that monocytes retained in the bone marrow of infected of infection and then continue in a programmed, inflammation- CCR2Ϫ/Ϫ mice were maturing (Fig. 6B). In contrast, significantly independent fashion. To address the role of ongoing infection in fewer Ly6ChighCD11bϩ/Ϫ and Sca-1highLy6Chigh cells were monopoiesis during L. monocytogenes infection, we studied the present in the bone marrows of antibiotic-treated CCR2Ϫ/Ϫ mice The Journal of Immunology 1907 Downloaded from http://www.jimmunol.org/

FIGURE 7. Monocyte reconstitution in MyD88/Trif knockout (KO) mice. Wild-type (WT) and MyD88Ϫ/ϪTriflps2/lps2 mice were infected with 1000 L. monocytogenes and spleens and femurs were harvested 2 and 3 days after infection. A, Splenocytes were stained for Ly6C and CD11b. The percentages of Ly6ChighCD11bϩ (monocytes) and Ly6CintCD11bhigh (granulocytes) cells are indicated above each gate. Representative dot plots for two to three mice ϩ ϩ per group are shown. B, Total numbers of Ly6ChighCD11b and Ly6G cells in the spleens. Each bar represents two to three mice (error bars, SD). C, by guest on September 25, 2021 Bone marrow cells were harvested at the indicated times and expression of Sca-1, Ly6C, and CD11b was analyzed. D, Total numbers of Ly6ChighCCR2ϩ and Ly6CintCD11bhigh cells in the bone marrow of infected and naive mice. Each bar represents two to three mice (error bars, SD). This experiment was repeated three times with similar results. The unpaired Student t test was used to compare groups. Statistical analysis was performed with Prism (GraphPad Software).

(Fig. 6, B and C). Thus, ampicillin treatment, by curtailing naling, responses in mice doubly deficient in adaptor molecules L. monocytogenes infection, abrogated accumulation of Ly6Chigh MyD88 and Trif were examined. Wild-type and MyD88Ϫ/Ϫ CD11bϩ cells in the bone marrow (Fig. 6, A and C). Triflps2/lps2 mice were infected with L. monocytogenes and mono- The proportion of BrdUϩLy6Chigh cells was significantly cyte composition in the spleens was examined 2 and 3 days after higher and the total number of proliferating Ly6Chigh cells was infection. Because of significant susceptibility of MyD88Ϫ/Ϫ increased Ͼ2-fold in infected CCR2-deficient mice compared Triflps2/lps2 mice to infection, a lower dose of 1000 Listeria was with antibiotic-treated and naive mice (Fig. 6, D and E). In administered in these experiments. As reported previously (6), the naive and antibiotic-treated mice, BrdUϩ cells were comprised percentages of splenic Ly6ChighCD11bϩ monocytes were similar largely of nonmyeloid (Ly6CϪCD11bϪ) and Ly6Cint cells. In at day 2 in both groups of infected mice, suggesting that monocyte contrast, the majority of proliferating cells in infected mice emigration is not impaired in the absence of TLR signaling were promonocytes and monocytes (Fig. 6D). Thus, while total (Fig. 7A). However, a paucity of monocytes was observed in the bone marrow cellularity and the number of proliferating non- spleens of MyD88Ϫ/ϪTriflps2/lps2 mice 3 days after infection monocytic populations were equally reduced in untreated and (Fig. 7A). Moreover, a 3- to 4-fold reduction in the numbers Ϫ Ϫ antibiotic-treated L. monocytogenes-infected CCR2 / mice, of Ly6ChighCD11bϩ monocytes was observed in spleens of subsequent monocyte expansion and differentiation was abro- MyD88Ϫ/ϪTriflps2/lps2 animals (Fig. 7B). In contrast, the fre- gated by antibiotic treatment, suggesting that ongoing infection quencies and total numbers of neutrophils (Ly6CintCD11bϩ)in drives this process. Moreover, accumulation of mature mono- the spleens of wild-type and MyD88Ϫ/ϪTriflps2/lps2 mice were cytes in the bone marrow did not suppress monocyte regener- comparable 2 and 3 days following L. monocytogenes infection ation, suggesting that monocytopenia was not required for this (Fig. 7, A and B). process. We then examined whether the reduction in the numbers of splenic Ly6ChighCD11bϩ and CCR2ϩ cells in the absence of Role of TLR signaling in monopoiesis TLR signaling was due to impaired monocyte replenishment in We next asked whether inflammation-driven monopoiesis required the bone marrow. In the absence of infection, the numbers of TLR-mediated signaling. To ensure complete absence of TLR sig- monocytes and neutrophils were comparable in wild-type and 1908 MONOPOIESIS DURING MICROBIAL INFECTION

MyD88Ϫ/ϪTriflps2/lps2 mice, indicating that MyD88/Trif signal- ing is not required for the development of these subsets during homeostasis (Fig. 7D). Comparable reduction in the total num- bers of bone marrow cells was observed in both groups of mice (data not shown). Both 2 and 3 days following infection, the percentage of bone marrow Sca-1highLy6Chigh cells was re- duced in MyD88Ϫ/ϪTriflps2/lps2 compared with wild-type mice (Fig. 7C). Moreover, the percentage of Sca-1highCD11bϩ monocytes was drastically reduced in the bone marrow of knockout mice at these times (Fig. 7C). Absence of Sca-1high precursor cells in the bone marrow led to dramatic depletion of bone marrow CCR2ϩ cells (Fig. 7D), suggesting that inflam- mation-driven monopoiesis is impaired in the absence of TLR signaling. In contrast, the numbers of neutrophils were similar between both groups of mice at all time points examined (Fig. 7D). To examine whether TLR signaling directly impacts prolifera- tion of monocyte precursors, we compared BrdU incorporation by Ϫ Ϫ bone marrow monocytes in wild-type and MyD88 / Triflps2/lps2 Downloaded from mice. Under homeostatic conditions, equal rates of proliferation were observed in wild-type and MyD88Ϫ/ϪTriflps2/lps2 monocytes (Fig. 8A). An overall reduction in the numbers of BrdUϩ cells was observed between days 2 and 3 after infection in both groups of mice and the total numbers of BrdUϩ cells were similar in both

groups (Fig. 8B). In wild-type bone marrow, frequencies of http://www.jimmunol.org/ CD115ϩ cells and CCR2ϩ cells were increased within the BrdUϩ population by day 3 after infection (Fig. 8C). In contrast, BrdUϩ cells in MyD88Ϫ/ϪTriflps2/lps2 mice were largely devoid of CD115ϩCCR2ϩ monocytes 3 days following infection (Fig. 8C). Overall, the numbers of Ly6ChighBrdUϩ, Sca-1highBrdUϩ, and CCR2ϩBrdUϩ cells were significantly reduced in the bone mar- row of knockout mice 3 days after infection compared with the wild-type mice (Fig. 8D). To confirm that the effect of TLR sig- naling was specific to monocytes, proliferation of neutrophil pro- by guest on September 25, 2021 genitors was also examined. Incorporation of BrdU by Ly6Cint granulocytes was similar in both groups of mice (Fig. 8D), indi- cating that deficiency in the MyD88/Trif pathway did not impact proliferation by this subset. To rule out contributions of IL-1 and IL-18 during emergency monopoiesis, bone marrow monocytes were examined in mice deficient in IL-1␤-converting enzyme (ICE, caspase 1). Significant increases in the numbers of Ly6Chigh BrdUϩ cells in the bone marrow of ICEϪ/Ϫ mice were observed (data not shown), demonstrating that signaling through IL-1 and IL-18 was not required for monocyte replenishment in the bone marrow. FIGURE 8. Monocyte proliferation in the absence of TLR signaling. Wild-type (WT) and MyD88Ϫ/ϪTriflps2/lps2 mice were left uninfected or infected with 1500 L. monocytogenes and BrdU was administered 2 and 3 Discussion days after infection. A, Bone marrow cells from naive mice were stained Recruitment of neutrophils and monocytes to peripheral sites is for BrdU, Ly6C, and CD11b and the frequencies of BrdUϩ cells in the essential for defense against a number of microbial pathogens. Ly6ChighCD11bϩ (monocytes) gate were examined. B, Total numbers of Under homeostatic conditions, the frequency of these cells in pe- BrdUϩ cells in the femurs. Each time point represents two to three mice ripheral tissues is low. Following infection, their numbers increase (error bars, SD). C, Cells were gated on BrdU and expression of CD115 dramatically due, in part, to accelerated emigration from bone mar- and CCR2 by BrdUϩ cells was analyzed. Representative dot plots are row and, in part, to enhanced recruitment into inflamed tissues. shown with a total of four to six mice analyzed. D, Total numbers of ϩ high ϩ high ϩ ϩ int high Although recruitment and activation of myeloid cells into periph- BrdU Ly6C , BrdU Sca-1 , BrdU CCR2 , and Ly6C CD11b eral tissues has been studied, relatively little is known about in- cells in the femurs. Each time point represents two to three mice (error bars, SD) and the experiment was repeated two times. The unpaired Stu- flammation-driven bone marrow hematopoiesis. In this study, we dent t test was used to compare groups. Statistical analysis was performed have examined the dynamics of bone marrow myeloid populations with Prism (GraphPad Software). KO, Knockout. following infection with the intracellular bacterial pathogen L. monocytogenes. Our results indicate that inflammation induced by L. monocytogenes infection alters the hematopoietic compartment the bone marrow during later stages of L. monocytogenes infec- and specifically promotes monopoiesis. In contrast to the effect of tion. We conclude that proliferation and replenishment of mono- this infection on monopoiesis, granulocyte production in the bone cyte precursors is dependant on ongoing inflammation and requires marrow was diminished. In the absence of MyD88/Trif-mediated TLR-mediated signaling. Thus, innate immune deficiency result- signaling, proliferation of monocyte precursors was diminished in ing from loss of MyD88 signaling stems not only from inadequate The Journal of Immunology 1909 activation of recruited monocytes at sites of infection (6), but also Signaling through TLR ligands expressed on the surface of from impaired production of monocytes in the bone marrow. monocytes could potentially induce cells to enter the cell cycle. The cellular composition of the bone marrow is impacted by Although a large body of literature demonstrates the prominent inflammation (22, 23). Drastic reduction in total bone marrow cel- role of TLR signaling in inducing differentiation rather than pro- lularity is observed following viral infection (24). It has been dem- liferation of immature cells, a recent study by Nagai et al. (28) onstrated that adjuvant administration and infection leads to estab- reported that stimulation with TLR ligands induced lineage-nega- lishment of bone marrow lymphopenia due to depletion of both B tive monocyte progenitors to enter the cell cycle. Thus, it is pos- cell progenitors and mature B cells (23–25) in a manner that is at sible that sustained monocyte proliferation following L. monocy- least partially dependent on TNF-␣ and IL-1 (23). It has been togenes infection is a direct result of engagement of TLRs on the proposed that lymphocytes and granulocytes share a common surface of monoblasts and promonocytes. Recently, several studies niche in the bone marrow and reduction in the numbers of lym- reported that mammalian microRNAs (miRNA) are involved in phoid precursors due to diminished levels of growth factors may the regulation of hematopoiesis and monopoiesis and could act lead to a compensatory increase in granulopoiesis (22). The results through regulation of expression of lineage-specific factors such as of our study suggest that monocyte expansion in the bone marrow M-CSF (32–34). Interestingly, expression of miRNAs can be in- is controlled in a manner distinct from granulopoiesis and that duced in bone marrow following administration of LPS (33). It increased availability of bone marrow niches or growth factors will be of interest to examine whether MyD88/Trif-dependent mo- may not be an underlying cause of monopoiesis. Our reasoning is nopoiesis during infection involves coordinate expression of based on several observations. Administration of antibiotics to in- miRNAs. Following i.v. administration of L. monocytogenes, bone fected mice significantly reduced proliferation of monocyte pro- marrow becomes infected and bacteria persist at this site for sev- Downloaded from genitors despite significant reduction in total bone marrow cellu- eral days (11). TLR-driven monocyte expansion may require direct larity. Likewise, a paucity of monocytes in the absence of MyD88/ presence of bacteria in the bone marrow or, alternatively, may Trif signaling occurred despite normal reductions in bone marrow result from circulating bacterial products. Because bone marrow cell numbers following infection. Thus, we believe that it is un- infection occurs concordantly with infection of peripheral organs, likely that perturbations in the numbers of cells of other lineages we cannot distinguish between these possibilities.

are the cause of enhanced . In addition to direct signaling in monocytes, TLR ligands may http://www.jimmunol.org/ Under noninflammatory conditions, M-CSF is required for both activate other cell types essential for induction of monopoiesis proliferation of promonocytes and survival of mature monocytes/ during inflammation. Under steady-state conditions, bone marrow macrophages. Selective destruction of M-CSF by differentiated dendritic cells are essential for survival of resident bone marrow B monocytes has been suggested to control in vivo macrophage pro- cells and for survival of recirculating B cells (35). Interestingly, duction (26, 27). Because significant efflux of monocytes from the MyD88 regulates development of , cells of monocyte- bone marrow is observed during the first 2 days following infec- macrophage lineage, in response to LPS, diacyl peptide, and IL-1␣ tion, subsequent freeing of proliferative niches and/or increased in vitro (36). TLR ligand-driven osteoclastogenesis involves availability of monocyte growth factors could drive expansion of MyD88-dependent induction of RANKL expression in osteoblasts. Ly6Chigh cells. However, our finding that monocyte expansion is In conclusion, we demonstrate that bacterial infection leads to by guest on September 25, 2021 observed in CCR2-deficient mice concurrent with accumulation of restructuring of the hematopoietic compartment and induces pref- mature monocytes argues against this hypothesis. erential expansion of monocytes in the bone marrow. MyD88/Trif An alternative explanation of our findings is that monopoiesis is signaling is essential for this process and MyD88/Trif-deficient driven directly by inflammatory cues. The recent demonstration mice experience monocytopenia following the onset of infection. that myeloid progenitors express TLR2 and TLR4 and are driven Previous studies have addressed the role of TLR- and MyD88- to proliferate and differentiate following TLR ligation in vitro (28) mediated signaling in the activation of monocytes. To our knowl- suggested that TLR signaling may be implicated in the regulation edge, we provide the first demonstration in an in vivo model of of hematopoiesis. Indeed, lymphoid progenitors express TLR9 and microbial infection that MyD88 signaling is involved in the re- are skewed toward dendritic cell differentiation following CpG ad- plenishment of monocytes. Additional experiments are required to ministration or murine HSV-1 infection (29). These findings sug- address the mechanisms of TLR-directed monopoiesis. gested the intriguing possibility that TLR signaling in the bone marrow may enlarge the pool of immature myeloid progenitors Disclosures during infection. However, our findings do not support this sce- The authors have no financial conflict of interest. nario and suggest that microbial infection instead leads to a sig- nificant depletion of lineage-negative myeloid progenitors. The mechanisms underlying loss of lineage-negative progenitors are References 1. van Furth, R., and Z. A. Cohn. 1968. The origin and kinetics of mononuclear currently not known. One explanation is that infection drives en- . J. Exp. Med. 128: 415–435. hanced maturation of these cells toward more committed popula- 2. Gordon, S., and P. R. Taylor. 2005. Monocyte and macrophage heterogeneity. tions, in the process depleting the early progenitor pool. Alterna- Nat. Rev. Immunol. 5: 953–964. 3. Serbina, N. V., T. Jia, T. M. Hohl, and E. G. 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