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CCR2-mediated recruitment of Ly6Chigh monocytes is essential for defense against a range of microbial pathogens. Although our understanding of monocyte 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 bone marrow 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 demonstrate that TLR-mediated signals play an essential role in the maintenance of monocyte homeostasis during systemic bacterial infection.
Monocytes arise from myeloid progenitors in the bone marrow and are released into the peripheral circulation (1). Bloodstream monocytes are heterogeneous and distinct subsets can be defined by expression of CD11b, Ly6C, 7/4, F4/80, and several chemokine receptors (2). The CCR2 chemokine receptor is expressed on a subset of circulating monocytes that traffic to sites of inflammation. CCR2+ monocyte trafficking is induced by some infections and can be essential for protection against intracellular bacterial pathogens, including Listeria monocytogenes (3).
L. monocytogenes is a Gram-positive facultative intracellular bacterium that infects a wide range of invertebrate and vertebrate hosts. Originally identified as a causative agent of a lethal disease in rabbits, it was noted to induce marked monocytosis in infected animals; hence its name. Murine infection with L. monocytogenes has been used extensively as a model for studying immune responses to intracellular bacterial pathogens. Innate immunity is rapidly triggered following infection and is crucial for initial control of bacterial replication. Following invasion of mammalian cells, L. monocytogenes initiates escape from the vacuole by secreting listeriolysin O, an essential virulence factor (4). Escape of L. monocytogenes to the cytosol is required for the induction of proinflammatory mediators and establishment of protective immunity. Secretion of MCP-1 and MCP-3, two chemokines that signal via CCR2, requires cytoplasmic invasion by bacteria and does not occur when heat-killed or noninvasive bacteria are administered to mice (5, 6).
During systemic infection with L. monocytogenes, CCR2+ monocytes are recruited to the spleen and differentiate into TNF- and inducible NO synthase (iNOS)4-producing dendritic cells (7) in a process that depends on dendritic cells and NK cells (8). TNF- and iNOS-producing dendritic cells are the major source of TNF and iNOS during L. monocytogenes infection and failure to recruit these cells leads to unrestricted bacterial replication and impaired host survival. Recent studies demonstrated recruitment of TNF- and iNOS-producing monocytes to gut-associated tissues following infection with Salmonella typhimurium or Toxoplasma gondii (9, 10), and CCR2-dependent monocyte recruitment is critical for control of T. gondii infection (9). Thus, CCR2+ monocytes may play a more general role in innate immune responses to microbial infections.
Following infection with L. monocytogenes, CCR2-mediated signals promote monocyte exit from bone marrow to the peripheral circulation. Emigration of mature monocytes from the bone marrow is more rapid than monocyte replenishment from progenitors, resulting, during the first 24 – 48 h of infection, in markedly diminished monocyte numbers in the bone marrow (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-negative bone marrow progenitors referred to as macrophage and dendritic cell progenitors (MDPs), which express c-kit (receptor for stem cell factor) and chemokine receptor CX3CR1 (12). This subset also expresses CD115 (M-CSF receptor) and can express receptor tyrosine kinase Flt3 (Flk2, CD135) (13). The fate of MDPs and their progeny following infection has not been examined.
In this study, we report that monocytes are continuously recruited to the spleens during the course of L. monocytogenes infection. Monocyte-mediated immunity is comprised of two distinct phases. In the first phase, significant numbers of pre-existing monocytes are recruited from the bone marrow to the periphery in response to infection. During the second phase, infection of mice results in rapid, robust but selective expansion of monocyte precursors in the bone marrow, leading to continuous replenishment of monocytes in the periphery. Monocyte expansion is not due to general expansion of the myeloid compartment because the number of bone marrow granulocytes is concurrently reduced. Infection-induced monopoiesis requires MyD88-dependent and IL-1/IL-18- independent signaling. These results suggest that inflammation induced by L. monocytogenes infection redirects the development of individual hematopoietic lineages in bone marrow by specifically promoting monopoiesis to sustain delivery of monocytes to peripheral sites of infection.
C57BL/6 and CCR2−/− mice were bred at Memorial Sloan-Kettering Research Animal Resource Center. Triflps2/lps2 mice were a gift from Dr. B. Beutler (The Scripps Research Institute, University of California, San Diego, CA) and bred in-house to MyD88−/− mice obtained from Dr. S. Akira (University of Osaka, Osaka, Japan). All mice were backcrossed at least 10 generations onto the C57BL/6 background. ICE−/− on the NOD/ShiLt background and BALB/cJ mice were purchased from The Jackson Laboratory. Mice were infected i.v. with 3000 L. monocytogenes strain 10403S (provided by Dr. D. Portnoy, University of California, Berkeley, CA). For infection of MyD88−/−Triflps2/lps2 mice and corresponding controls, a lower dose of 1000 L. monocytogenes was used. When indicated, ampicillin (Sigma-Aldrich) was given to mice at 2 mg/ml in drinking water. All animal experiments were approved by the Institutional Animal Care and Use Committee.
CCR2 reporter mice were generated by bacterial artificial chromosome (BAC)-mediated transgenesis using the recombineering strategy pioneered by Heintz and colleagues (14). Briefly, the endogenous CCR2 locus was identified on BAC clone RP23-182D4 (~250 kb in size; obtained from CHORI) derived from the distal end of chromosome 9 by PCR screening of candidate BACs mapped to this region. A 1.7-kb fragment that contained 1.0 kb upstream and 0.7 kb downstream of the CCR2 gene start codon was amplified and modified between the first and second codon of CCR2 through insertion of nucleotides that encoded aa 2–238 of enhanced GFP. The GFP coding sequence was followed by a stop codon and the ensuing nucleotide at position 4 of the endogenous CCR2 locus was deleted. The resulting 2.4-kb fragment was cloned into the shuttle vector pLD53SC.AB obtained from Dr. D. Littman (New York University, New York, NY) (14).
To modify BAC clone RP23-182D4 for transgenesis, the shuttle vector containing the 2.4-kb CCR2.GFP insert was integrated into the BAC by homologous recombination and cointegrates were identified by chloramphenicol and ampicillin selection. Resolution of cointegrates through a second homologous recombination event was achieved by negative selection on sucrose, resulting in the complete excision of the shuttle vector backbone that includes the SacB gene (for details, see Ref. 14). The resulting modified BAC encoding GFP under control of the endogenous CCR2 promoter and regulatory elements was analyzed by Southern blotting to verify GFP integration at the expected site, sequenced over the CCR2 gene locus, purified by centrifugation through a cesium chloride gradient, and injected into fertilized C57BL/6J oocytes. Four potential founder animals were identified among 39 off-spring screened by PCR and flow cytometric analysis of peripheral blood samples.
One candidate, designated CCR2.GFP.8, transmitted the transgene to one-half of the progeny and was the founder of the CCR2 reporter colony. CCR2 reporter mice were maintained under specific pathogen-free conditions on standard laboratory chow. For specific experiments, the CCR2 reporter mice were bred to MyD88−/−Triflps2/lps2 mice on the C57BL/6J background. CCR2 reporter MyD88−/−Triflps2/lps2 animals were maintained on Sulfatrim feed before use in experiments.
Spleens were harvested at indicated times postinfection, dissociated, and digested with 0.3% collagenase type 4 (Worthington Biochemical). Bone marrow cells were harvested from mouse femurs. Erythrocytes were lysed with NH4Cl-Tris solution and cells were stained for cell surface and intracellular markers. The following Abs were purchased from BD Pharmingen: anti-CD11b (M1/70), anti-CD11c (HL3), anti-B7-1 (16-10A1), anti-B7-2 (GL1), anti-I-A/I-E (M5/114.15.2), anti-Ly6G (1A8), anti-Ly6C (AL-21), anti-c-kit (104D2), anti-Sca-1 (D7), and anti-Flk2 (A2F10.1). Anti-CD115 (AFS98) was purchased from eBioscience. Anti-F40/80 (A3-1) and anti-monocyte/neutrophil (7/4) were purchased from Serotec. The lineage Ab mixture included the following Abs (BD Pharmingen): anti-LyC and Ly6G (RB6-8C5), anti-CD11b (M1/70), anti-CD3e (145-2C11), anti-CD45/B220 (RA3-6B2), anti-erythroid cells (TER-119), and anti-NK 1.1 (PK136). For morphological examination, cell subsets were obtained by fluorescence-activated sorting with the purity of >98%. Cells were spun onto glass slides and stained using Diff-Quik Satin Set (Dade Behring).
Sca-1+Ly6C−, Sca-1+Ly6C+CD11blow, and Sca-1+Ly6C+CD11bhigh cells were sorted from bone marrows of wild-type mice infected for 3 days. Sorted cells were plated at 105 cells/well in 96-well, flat-bottom plates in DMEM (Life Technologies) supplemented with 10% FCS, l-glutamine, HEPES, 2-ME, antibiotics (penicillin, streptomycin, gentamicin) and recombinant growth factors (R&D Systems), and cells were cultured for 72 h. The resultant cultures were harvested and analyzed by flow cytometry.
In vivo BrdU labeling and staining was conducted using BrdU Flow Kits from BD Pharmingen. Briefly, mice were administered 1 mg of BrdU solution via the i.p. route and femurs were collected 1–2 h later. Single-cell bone marrow suspensions were prepared and processed for cell surface Ag staining followed by staining for incorporated BrdU according to the manufacturer's protocol.
Bone marrow monocytes are characterized by high levels of Ly6C and intermediate levels of CD11b expression. Under homeostatic conditions, Ly6Chigh monocytes represent 5–8% of the total nucleated bone marrow cells and are ~5-fold less frequent than Ly6G+ neutrophils (35–50% of bone marrow cells; data not shown). In uninfected mice, Ly6Chigh monocytes express 7/4, M-CSF receptor CD115, and do not express the granulocyte marker Ly6G (Fig. 1A). Additionally, Ly6Chigh bone marrow monocytes are immature and do not express MHC class II, costimulatory molecules, or CD11c (Fig. 1A). Bone marrow monocytes express the chemokine receptor CCR2 and require CCR2-mediated signals for bone marrow egress during infection (11). To facilitate detection of CCR2-expressing monocytes, we generated transgenic mice in which GFP expression is controlled by the CCR2 promoter. In these CCR2 reporter mice, Ly6Chigh monocytes are uniformly labeled with GFP, confirming that they represent a homogeneous population. Sorted Ly6Chigh monocytes are of uniform morphology (Fig. 1A).
L. monocytogenes infection induces recruitment of Ly6Chigh monocytes from the bone marrow to sites of infection (7). To determine the extent of infection-induced bone marrow monocyte depletion, bone marrow cellularity was measured at various times following systemic L. monocytogenes infection. Total numbers of bone marrow cells in infected mice declined between days 1 and 4 after infection (Fig. 1B) and the percentage of Ly6Chigh monocytes declined for the first 2 days following infection. Three and 4 days after infection, however, the frequency and total number of Ly6Chigh monocytes increased (Fig. 1, C and D). In contrast to the increasing number of bone marrow monocytes, the total number of granulocytes in bone marrow remained low for the first 4 days of systemic infection (Fig. 1E), suggesting that monopoiesis but not granulopoiesis was induced by L. monocytogenes infection. Because monocytes are continuously emigrating from bone marrow to peripheral sites during infection, the static measure of bone marrow cellularity likely underestimates the number of newly generated monocytes. Indeed, marked monocyte recruitment to the spleen was observed between days 2 and 4 after infection (Fig. 1F), leading to dramatic increases in the total number of splenic monocytes (Fig. 1G). In contrast, peak granulocyte recruitment to the spleen was detected on the second day of infection and subsequently decreased (Fig. 1H).
We reasoned that the increase in the numbers of bone marrow Ly6Chigh monocytes following infection might result from an expansion of lineage-negative progenitors committed to the monocyte/macrophage lineage. The total number of lineage- negative cells in the bone marrow of L. monocytogenes-infected mice, however, remained stable for the first 2 days after infection and then rapidly declined (Fig. 2A). We next examined the frequencies of lineage-negative bone marrow progenitors that can give rise to monocytes. In the bone marrow, multipotent progenitors (c-kit+Sca-1+CD127−FcγR−) give rise to common myeloid progenitors (c-kit+ Sca-1−CD127−FcγR−) which further differentiate into granulocyte/macrophage progenitors (c-kit+Sca-1−CD127−FcγR+) (15). A recently identified subset of progenitor cells expressing c-kit, CD115. and CX3CR1 (MDPs) (12, 13) can differentiate into monocytes, macrophages, and dendritic cells but not granulocytes and is derived from granulocyte/macrophage progenitors. In the uninfected bone marrow, c-kit+CD115+ cells represented ~25% of the lineage-negative population (Fig. 2B). These cells expressed FcγR and did not express CD127 (data not shown) and thus phenotypically resembled MDPs (16, 17). Surprisingly, expression of CD115 in the Lin−c-kit+ populations diminished dramatically between days 1 and 2 following infection and CD115+ cells reappeared only at day 4 (Fig. 2B). Overall, we observed a drastic decrease in the number of c-kit+CD115+ monocyte progenitors following infection (Fig. 2C). Because monocytic progenitors also express tyrosine kinase Flk2 (Flt3) (12, 18), we additionally examined the presence of Lin−c-kit+Flk2+ cells in the bone marrow at various times after infection. We found that the number of these cells was also significantly reduced between days 1 and 4 (data not shown). Thus, inflammation did not lead to a rapid replenishment of the immature progenitor pool.
Under homeostatic conditions, mature monocytes are the nonproliferating progeny of dividing monoblasts and promonocytes. We next examined whether Ly6Chigh cells were actively dividing during the course of infection. To assess cellular proliferation, BrdU was administered to mice at distinct times following infection and bone marrow cells were collected 1-2 h later. The short duration of BrdU pulse allowed us to estimate the proportion of cells entering and progressing through the S phase of the cell cycle at the time of bone marrow harvest. In uninfected mice and in mice infected for 1 day, the majority of DNA-synthesizing cells were Ly6C− and Ly6Chigh cells represented only a small fraction of these cells (Fig. 3A). By day 2 of infection, the proportion of Ly6Chigh cells increased within the BrdU+ pool and a concomitant decrease was observed in proliferating Ly6C− cells. As infection progressed, the incorporation of BrdU was predominantly in Ly6Chigh cells, and these cells accounted for ~70% of the total cells undergoing DNA synthesis (Fig. 3A and data not shown). Within the Ly6Chigh population, a significantly higher fraction of cells was driven to divide by day 3 following infection (Fig. 3B). The number of Ly6Chigh cells that incorporated BrdU increased significantly between days 1 and 3 after infection (Fig. 3C). Although most BrdU+ cells on day 2 of infection were Ly6Chigh CD11b−, by day 3 the BrdU+ population was comprised of both CD11b− and CD11b+ populations (Fig. 3C). At the same time, the number of proliferating Ly6Cint cells (granulocytes) decreased (Fig. 3C). Thus, L. monocytogenes infection specifically promotes expansion of monocytes while suppressing proliferation of other cell lineages.
Although bacterial infection decreases the frequency of lineage-negative monocyte progenitors in bone marrow, the effect on the frequency of later stage monocyte progenitors and monocyte maturation is unknown. We reasoned that newly replenished monocytes may retain expression of some markers of lineage-negative monocyte precursors. Because Sca-1 (Ly6 A/E) and c-kit have both been useful markers to distinguish bone marrow stem cells and progenitors, we chose to examine the expression of these Ags on bone marrow populations. We therefore investigated monocyte differentiation by characterizing co-expression of lineage markers and either c-kit or Sca-1 on bone marrow cells 1, 2, and 3 days following systemic L. monocytogenes infection. Although expression of lineage markers was associated with down-modulation of c-kit (data not shown), we detected a dramatic increase in the frequency of Sca-1highLin+ cells in the bone marrow of mice 2 and 3 days after infection (Fig. 4A). We next characterized the Sca-1high population in greater detail. In naive mice and in mice 1 day following infection, very few Sca-1high cells were present in the bone marrow (Fig. 4B and data not shown). The percentage of Sca-1high cells increased 2 and 3 days after infection and most of these cells expressed high levels of Ly6C (Fig. 4B). Only a fraction of Sca-1highLy6Chigh cells, however, expressed CD11b at days 2 and 3 after infection (Fig. 4C). To determine whether Sca-1highLy6Chigh cells represent inflammatory monocyte progenitors, expression of CCR2, CD115, and CD11b by this population was further examined. At day 3 after infection, the majority of Sca-1highLy6Chigh cells expressed CCR2 and CD115 and heterogeneous levels of CD11b (Fig. 4D). To confirm that Sca-1high cells represent newly replenishing monocytes, proliferation of these cells was also examined. We observed significant BrdU incorporation by Sca-1high cells 3 days following infection and the percentage of Ly6ChighBrdU+ cells correlated closely with the percentage of Sca-1highBrdU+ cells (data not shown).
Sca-1highLy6Chigh CD11b−/low and Sca-1highLy6ChighCD11b+ cells were sorted from bone marrow 2 days after infection for morphological analysis. Sca-1highLy6ChighCD11b− cells were small round cells with a small rim of basophilic cytoplasm, few cytoplasmic vesicles, and a >1 ratio of nucleus:cytoplasm. This population also contained cells with ring-shaped nuclei with a karyoplasmic ring wider than the diameter of its center, a morphology that is characteristic of the mononuclear lineage (19) (Fig. 4E). Sca-1highLy6ChighCD11b+ cells were larger in size with less basophilic cytoplasm, more abundant vacuoles. and some pseudopods. The nucleus:cytoplasm ratio in this cell population was ~1 or >1 and nuclei had a horseshoe or kidney shape (Fig. 4E). Thus, Sca-1highLy6ChighCD11b+ cells represent populations of immature monocytes while Sca-1highLy6Chigh CD11b− cells are monoblasts.
The up-regulation of Ly6C and CD11b expression by Sca-1high cells could result from sequential differentiation of Ly6C−CD11b− cells toward Ly6ChighCD11b− and then toward Ly6ChighCD11b+ cells. Alternatively, these cells may represent distinct differentiation branches that are not linearly related. To determine the potential of Sca-1highLy6C−CD11b− and Sca-1highLy6ChighCD11b− cells to differentiate into CD11b-expressing monocytes/macrophages, we sorted Sca-1highLy6C−CD11b−, Sca-1highLy6ChighCD11b−, and Sca-1highLy6Chigh CD11b+ cells from bone marrow of mice infected for 2 days with L. monocytogenes. Sorted cells were cultured in vitro for 24–48 h in the presence of M-CSF or GM-CSF. Culture in the presence of either growth factor led to survival of all sorted cell populations with the best recovery observed for the Sca-1highLy6C+CD11b− population (Fig. 5A). Cultures of Sca-1highLy6C− cells were characterized by small forward scatter properties characteristic of lymphocytes while cells derived from Sca-1highLy6ChighCD11b− and Sca-1highLy6ChighCD11b+ populations were significantly larger (Fig. 5B). Despite enhanced survival of Sca-1highLy6C− cells in the presence of M-CSF and GM-CSF, very few Ly6ChighCD11b+F4/80+ cells could be recovered from resultant cultures (<5%), suggesting that Sca-1highLy6C− cells did not have in vitro macrophage differentiation potential (Fig. 5C). In contrast, cultures of Sca-1highLy6Chigh CD11b− and Sca-1highLy6Chigh CD11b+ cells yielded a similar frequency of CD11b+F4/80+ cells, suggesting that Sca-1highLy6ChighCD11b− cells can differentiate toward CD11b-expressing macrophages. Thus, the bone marrow population of Sca-1highLy6ChighCD11b− cells represents monoblast progenitors of monocytes.
It is possible that monocyte proliferation and differentiation require continuous inflammation or progressing infection. Alternatively, bone marrow monopoiesis may be stimulated during early stages of infection and then continue in a programmed, inflammation-independent fashion. To address the role of ongoing infection in monopoiesis during L. monocytogenes infection, we studied the bone marrow of infected CCR2-deficient mice because monocyte emigration to the periphery is impaired in these mice (11, 20). This enabled us to measure monocyte expansion in bone marrow without the potential confounding effect of unmeasured monocyte emigration during the course of infection.
To directly address whether progressive infection is required for monopoiesis, infected mice were treated with oral ampicillin 24 h after initiation of infection. Treatment with ampicillin leads to bacterial clearance and reduction of infection-associated inflammation within 12 h of administration (21). Antibiotic administration did not interfere with the initial phase of cellular emigration from the bone marrow since comparable reductions in total cell numbers were observed in antibiotic-treated and untreated mice (Fig. 6A). Three days after infection of CCR2−/− mice, Sca-1highLy6Chigh and Ly6ChighCD11b+/− cells represented ~50% of the bone marrow population (Fig. 6B) and their numbers were significantly increased compared with naive controls (Fig. 6C and data not shown). Newly generated Ly6ChighCD11b+/− cells expressed CD115 and a significant proportion of these cells expressed F4/80, indicating that monocytes retained in the bone marrow of infected CCR2−/− mice were maturing (Fig. 6B). In contrast, significantly fewer Ly6ChighCD11b+/− and Sca-1highLy6Chigh cells were present in the bone marrows of antibiotic-treated CCR2−/− mice (Fig. 6, B and C). Thus, ampicillin treatment, by curtailing L. monocytogenes infection, abrogated accumulation of Ly6Chigh CD11b+ cells in the bone marrow (Fig. 6, A and C).
The proportion of BrdU+Ly6Chigh cells was significantly higher and the total number of proliferating Ly6Chigh cells was increased >2-fold in infected CCR2-deficient mice compared with antibiotic-treated and naive mice (Fig. 6, D and E). In naive and antibiotic-treated mice, BrdU+ cells were comprised largely of nonmyeloid (Ly6C−CD11b−) and Ly6Cint cells. In contrast, the majority of proliferating cells in infected mice were promonocytes and monocytes (Fig. 6D). Thus, while total bone marrow cellularity and the number of proliferating nonmonocytic populations were equally reduced in untreated and antibiotic-treated L. monocytogenes-infected CCR2−/− mice, subsequent monocyte expansion and differentiation was abrogated by antibiotic treatment, suggesting that ongoing infection drives this process. Moreover, accumulation of mature monocytes in the bone marrow did not suppress monocyte regeneration, suggesting that monocytopenia was not required for this process.
We next asked whether inflammation-driven monopoiesis required TLR-mediated signaling. To ensure complete absence of TLR signaling, responses in mice doubly deficient in adaptor molecules MyD88 and Trif were examined. Wild-type and MyD88−/− Triflps2/lps2 mice were infected with L. monocytogenes and monocyte composition in the spleens was examined 2 and 3 days after infection. Because of significant susceptibility of MyD88−/− Triflps2/lps2 mice to infection, a lower dose of 1000 Listeria was administered in these experiments. As reported previously (6), the percentages of splenic Ly6ChighCD11b+ monocytes were similar at day 2 in both groups of infected mice, suggesting that monocyte emigration is not impaired in the absence of TLR signaling (Fig. 7A). However, a paucity of monocytes was observed in the spleens of MyD88−/−Triflps2/lps2 mice 3 days after infection (Fig. 7A). Moreover, a 3- to 4-fold reduction in the numbers of Ly6ChighCD11b+ monocytes was observed in spleens of MyD88−/−Triflps2/lps2 animals (Fig. 7B). In contrast, the frequencies and total numbers of neutrophils (Ly6CintCD11b+) in the spleens of wild-type and MyD88−/−Triflps2/lps2 mice were comparable 2 and 3 days following L. monocytogenes infection (Fig. 7, A and B).
We then examined whether the reduction in the numbers of splenic Ly6ChighCD11b+ and CCR2+ cells in the absence of TLR signaling was due to impaired monocyte replenishment in the bone marrow. In the absence of infection, the numbers of monocytes and neutrophils were comparable in wild-type and MyD88−/−Triflps2/lps2 mice, indicating that MyD88/Trif signaling is not required for the development of these subsets during homeostasis (Fig. 7D). Comparable reduction in the total numbers 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 reduced in MyD88−/−Triflps2/lps2 compared with wild-type mice (Fig. 7C). Moreover, the percentage of Sca-1highCD11b+ 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 CCR2+ cells (Fig. 7D), suggesting that inflammation-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 proliferation of monocyte precursors, we compared BrdU incorporation by bone marrow monocytes in wild-type and MyD88−/−Triflps2/lps2 mice. Under homeostatic conditions, equal rates of proliferation were observed in wild-type and MyD88−/−Triflps2/lps2 monocytes (Fig. 8A). An overall reduction in the numbers of BrdU+ cells was observed between days 2 and 3 after infection in both groups of mice and the total numbers of BrdU+ cells were similar in both groups (Fig. 8B). In wild-type bone marrow, frequencies of CD115+ cells and CCR2+ cells were increased within the BrdU+ population by day 3 after infection (Fig. 8C). In contrast, BrdU+ cells in MyD88−/−Triflps2/lps2 mice were largely devoid of CD115+CCR2+ monocytes 3 days following infection (Fig. 8C). Overall, the numbers of Ly6ChighBrdU+, Sca-1highBrdU+, and CCR2+BrdU+ cells were significantly reduced in the bone marrow of knockout mice 3 days after infection compared with the wild-type mice (Fig. 8D). To confirm that the effect of TLR signaling was specific to monocytes, proliferation of neutrophil progenitors was also examined. Incorporation of BrdU by Ly6Cint granulocytes was similar in both groups of mice (Fig. 8D), indicating 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-1β-converting enzyme (ICE, caspase 1). Significant increases in the numbers of Ly6Chigh BrdU+ cells in the bone marrow of ICE−/− 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.
Recruitment of neutrophils and monocytes to peripheral sites is essential for defense against a number of microbial pathogens. Under homeostatic conditions, the frequency of these cells in peripheral tissues is low. Following infection, their numbers increase dramatically due, in part, to accelerated emigration from bone marrow and, in part, to enhanced recruitment into inflamed tissues. Although recruitment and activation of myeloid cells into peripheral tissues has been studied, relatively little is known about inflammation-driven bone marrow hematopoiesis. In this study, we have examined the dynamics of bone marrow myeloid populations following infection with the intracellular bacterial pathogen L. monocytogenes. Our results indicate that inflammation induced by L. monocytogenes infection alters the hematopoietic compartment and specifically promotes monopoiesis. In contrast to the effect of this infection on monopoiesis, granulocyte production in the bone marrow was diminished. In the absence of MyD88/Trif-mediated signaling, proliferation of monocyte precursors was diminished in the bone marrow during later stages of L. monocytogenes infection. We conclude that proliferation and replenishment of monocyte precursors is dependant on ongoing inflammation and requires TLR-mediated signaling. Thus, innate immune deficiency resulting from loss of MyD88 signaling stems not only from inadequate activation of recruited monocytes at sites of infection (6), but also from impaired production of monocytes in the bone marrow.
The cellular composition of the bone marrow is impacted by inflammation (22, 23). Drastic reduction in total bone marrow cellularity is observed following viral infection (24). It has been demonstrated that adjuvant administration and infection leads to establishment of bone marrow lymphopenia due to depletion of both B cell progenitors and mature B cells (23-25) in a manner that is at least partially dependent on TNF-α and IL-1 (23). It has been proposed that lymphocytes and granulocytes share a common niche in the bone marrow and reduction in the numbers of lymphoid precursors due to diminished levels of growth factors may lead to a compensatory increase in granulopoiesis (22). The results of our study suggest that monocyte expansion in the bone marrow is controlled in a manner distinct from granulopoiesis and that increased availability of bone marrow niches or growth factors may not be an underlying cause of monopoiesis. Our reasoning is based on several observations. Administration of antibiotics to infected mice significantly reduced proliferation of monocyte progenitors despite significant reduction in total bone marrow cellularity. Likewise, a paucity of monocytes in the absence of MyD88/Trif signaling occurred despite normal reductions in bone marrow cell numbers following infection. Thus, we believe that it is unlikely that perturbations in the numbers of cells of other lineages are the cause of enhanced monocytopoiesis.
Under noninflammatory conditions, M-CSF is required for both proliferation of promonocytes and survival of mature monocytes/macrophages. Selective destruction of M-CSF by differentiated monocytes has been suggested to control in vivo macrophage production (26, 27). Because significant efflux of monocytes from the bone marrow is observed during the first 2 days following infection, subsequent freeing of proliferative niches and/or increased availability of monocyte growth factors could drive expansion of Ly6Chigh cells. However, our finding that monocyte expansion is observed in CCR2-deficient mice concurrent with accumulation of mature monocytes argues against this hypothesis.
An alternative explanation of our findings is that monopoiesis is driven directly by inflammatory cues. The recent demonstration that myeloid progenitors express TLR2 and TLR4 and are driven to proliferate and differentiate following TLR ligation in vitro (28) suggested that TLR signaling may be implicated in the regulation of hematopoiesis. Indeed, lymphoid progenitors express TLR9 and are skewed toward dendritic cell differentiation following CpG administration or murine HSV-1 infection (29). These findings suggested the intriguing possibility that TLR signaling in the bone marrow may enlarge the pool of immature myeloid progenitors during infection. However, our findings do not support this scenario and suggest that microbial infection instead leads to a significant depletion of lineage-negative myeloid progenitors. The mechanisms underlying loss of lineage-negative progenitors are currently not known. One explanation is that infection drives enhanced maturation of these cells toward more committed populations, in the process depleting the early progenitor pool. Alternatively, inflammatory signaling may induce mobilization of immature progenitors to the periphery. Hematopoietic progenitor cells express chemokine receptors and recirculate through the blood under homeostatic conditions (30). A recent study by the von Andrian group (31) suggested that recirculating hematopoietic cells are capable of myeloid differentiation in peripheral tissues in response to inflammatory stimuli. Further studies are required to determine whether L. monocytogenes infection leads to enhanced mobilization of bone marrow hematopoietic progenitor cells and whether these cells can contribute to immune defense at peripheral sites.
Signaling through TLR ligands expressed on the surface of monocytes could potentially induce cells to enter the cell cycle. Although a large body of literature demonstrates the prominent role of TLR signaling in inducing differentiation rather than proliferation of immature cells, a recent study by Nagai et al. (28) reported that stimulation with TLR ligands induced lineage-negative monocyte progenitors to enter the cell cycle. Thus, it is possible that sustained monocyte proliferation following L. monocytogenes infection is a direct result of engagement of TLRs on the surface of monoblasts and promonocytes. Recently, several studies reported that mammalian microRNAs (miRNA) are involved in the regulation of hematopoiesis and monopoiesis and could act through regulation of expression of lineage-specific factors such as M-CSF (32-34). Interestingly, expression of miRNAs can be induced in bone marrow following administration of LPS (33). It will be of interest to examine whether MyD88/Trif-dependent monopoiesis during infection involves coordinate expression of miRNAs. Following i.v. administration of L. monocytogenes, bone marrow becomes infected and bacteria persist at this site for several days (11). TLR-driven monocyte expansion may require direct presence of bacteria in the bone marrow or, alternatively, may result from circulating bacterial products. Because bone marrow infection occurs concordantly with infection of peripheral organs, we cannot distinguish between these possibilities.
In addition to direct signaling in monocytes, TLR ligands may activate other cell types essential for induction of monopoiesis during inflammation. Under steady-state conditions, bone marrow dendritic cells are essential for survival of resident bone marrow B cells and for survival of recirculating B cells (35). Interestingly, MyD88 regulates development of osteoclasts, cells of monocyte-macrophage lineage, in response to LPS, diacyl peptide, and IL-1α in vitro (36). TLR ligand-driven osteoclastogenesis involves MyD88-dependent induction of RANKL expression in osteoblasts.
In conclusion, we demonstrate that bacterial infection leads to restructuring of the hematopoietic compartment and induces preferential expansion of monocytes in the bone marrow. MyD88/Trif signaling is essential for this process and MyD88/Trif-deficient mice experience monocytopenia following the onset of infection. Previous studies have addressed the role of TLR- and MyD88-mediated signaling in the activation of monocytes. To our knowledge, we provide the first demonstration in an in vivo model of microbial infection that MyD88 signaling is involved in the replenishment of monocytes. Additional experiments are required to address the mechanisms of TLR-directed monopoiesis.
1The authors' research is supported by the National Institutes of Health (Grants R37 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.).
4Abbreviations used in this paper: iNOS, inducible NO synthase; MDP, macrophage and dendritic cell progenitor; BAC, bacterial artificial chromosome; miRNA, microRNA; ICE, IL-1β-converting enzyme.
Disclosures The authors have no financial conflict of interest.