Microbiota-derived compounds drive steady-state granulopoiesis via MyD88/TICAM signaling

Microbiota-Derived Compounds Drive Steady-State Granulopoiesis via MyD88/TICAM Signaling This information is current as of November 20, 2016.

Maria L. Balmer, Christian M. Schürch, Yasuyuki Saito, Markus B. Geuking, Hai Li, Miguelangel Cuenca, Larisa V. Kovtonyuk, Kathy D. McCoy, Siegfried Hapfelmeier, Adrian F. Ochsenbein, Markus G. Manz, Emma Slack and Andrew J. Macpherson

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 9650 Rockville Pike, Bethesda, MD 20814-3994. Copyright © 2014 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606.

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J Immunol 2014; 193:5273-5283; Prepublished online 10 October 2014; doi: 10.4049/jimmunol.1400762 http://www.jimmunol.org/content/193/10/5273

The Journal of Immunology

Microbiota-Derived Compounds Drive Steady-State Granulopoiesis via MyD88/TICAM Signaling Maria L. Balmer,* Christian M. Sch€ urch,† Yasuyuki Saito,‡ Markus B. Geuking,* x Hai Li,* Miguelangel Cuenca, Larisa V. Kovtonyuk,‡ Kathy D. McCoy,* Siegfried Hapfelmeier,x Adrian F. Ochsenbein,† Markus G. Manz,‡ Emma Slack,*,{ and Andrew J. Macpherson*

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n detection of wounding or infection, neutrophils are recruited and swarm within minutes (1–3). The extreme sensitivity of neutropenic patients to infection demon-

*Division of Gastroenterology, Department of Clinical Research, University Clinic for Visceral Surgery and Medicine, University of Bern, 3010 Bern, Switzerland; † Department of Clinical Research, Tumor Immunology, University of Bern, 3010 Bern, Switzerland; ‡Division of Hematology, University Hospital Zurich, 8091 Zurich, Switzerland; xInstitute for Infectious Diseases, University of Bern, 3010 Bern, Switzerland; and {Institute of Microbiology, Swiss Federal Institute of Technology Zurich, 8093 Zurich, Switzerland Received for publication March 26, 2014. Accepted for publication September 9, 2014. A.J.M. was supported by the Swiss National Science Foundation (Grants 310030124732 and 313600-123736), the Canadian Institutes of Health Research, and the Genaxen Foundation. M.L.B. was supported by Oncosuisse and the Swiss National Science Foundation (Grant 313600-123736/1). S.H. and K.D.M. received funding from the European Research Council under the European Union’s Seventh Framework Programme (FP/2007-2013)/European Research Council Grants 281785 and 281904, respectively. A.F.O. was supported by the Swiss National Science Foundation (Grant 133132), Oncosuisse, and the Bernische Krebsliga. C.M.S. was supported by the Gertrud-Hagmann-Stiftung f€ur Malignomforschung and the SwissLife Jubila¨umsstiftung. M.G.M. was supported by the Swiss National Science Foundation (Grant 310030_146528/1). E.S. (PZ00P3_136742) and M.B.G. were supported by an Ambizione fellowship from the Swiss National Science Foundation. Address correspondence and reprint requests to Prof. Andrew J. Macpherson or Dr. Emma Slack, Division of Gastroenterology, Department of Clinical Research, University Clinic for Visceral Surgery and Medicine, University of Bern, Murtenstrasse 35, CH-3010 Bern, Switzerland (A.J.M.) or Institute of Microbiology, HCI G 413, Vladimir-Prelog-Weg 1-5/10, CH-8093 Zurich, Switzerland (E.S.). E-mail addresses: [email protected] (A.J.M.) or [email protected] (E.S.) Abbreviations used in this article: BM, bone marrow; CLP, common lymphoid progenitor; GF, germ-free; GMP, granulocyte-monocyte progenitor; HSPC, hematopoietic stem/progenitor cell; IVC, individually ventilated cage; LB, Luria–Bertani (media or agar); LCM, low-complexity microbiota; LSK, lineage2, Sca1+, c-kit “high” cells; MEP, megakaryocyte-erythrocyte progenitor; RT, room temperature; SPF, specific pathogen-free. Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00 www.jimmunol.org/cgi/doi/10.4049/jimmunol.1400762

strates that this rapid response is essential for survival (4). However, production of sufficient neutrophils is a costly procedure. An adult human produces thousands of neutrophils per second, which during health predominantly senesce (5). The possibility to “tune” the levels of neutrophil production to the level of threat faced by the organism would therefore appear to be beneficial. It is well documented that during severe bacterial infection, a process referred to as “emergency myelopoiesis” is induced (5, 6). In this situation, a much greater proportion of multipotent progenitor cells are diverted into the myeloid lineage at the expense of lymphopoiesis. This process appears to require sensing of pathogen-associated molecular patterns in a TLR-dependent manner (7). It is further known that massive production of IFN-g can enhance the production of monocytes over granulocytes and can drive erythropoiesis to start at sites outside the bone marrow (BM) (8). However, increased production of mature granulocytes requires hours to days in the human system, and although this increased production is central to the later control and eradication of infection, the initial encounter with pathogenic or apathogenic bacteria requires the immediate action of the homeostatically maintained circulating and marginated granulocyte pools (1, 6). We and others have previously noticed that germ-free (GF) mice show delayed kinetics of clearance of bacteria given i.v. (9, 10). GF mice are known to have an immature mucosal immune system, including reduced secondary lymphoid tissues, lower levels of secretory IgA, and fewer intestinal plasma cells (11). In addition, GF mice have lower levels of serum Abs and increased susceptibility to infection with a number of bacterial pathogens, both in the intestinal tract and systemically (11). It has been observed previously that treatment of specific pathogen-free (SPF), but not GF mice, with the lipid A–binding antibiotic polymyxin B reduces the number of granulocyte-monocyte colonies formed during in vitro

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Neutropenia is probably the strongest known predisposition to infection with otherwise harmless environmental or microbiotaderived species. Because initial swarming of neutrophils at the site of infection occurs within minutes, rather than the hours required to induce “emergency granulopoiesis,” the relevance of having high numbers of these cells available at any one time is obvious. We observed that germ-free (GF) animals show delayed clearance of an apathogenic bacterium after systemic challenge. In this article, we show that the size of the bone marrow myeloid cell pool correlates strongly with the complexity of the intestinal microbiota. The effect of colonization can be recapitulated by transferring sterile heat-treated serum from colonized mice into GF wild-type mice. TLR signaling was essential for microbiota-driven myelopoiesis, as microbiota colonization or transferring serum from colonized animals had no effect in GF MyD882/2TICAM12/2 mice. Amplification of myelopoiesis occurred in the absence of microbiota-specific IgG production. Thus, very low concentrations of microbial Ags and TLR ligands, well below the threshold required for induction of adaptive immunity, sets the bone marrow myeloid cell pool size. Coevolution of mammals with their microbiota has probably led to a reliance on microbiota-derived signals to provide tonic stimulation to the systemic innate immune system and to maintain vigilance to infection. This suggests that microbiota changes observed in dysbiosis, obesity, or antibiotic therapy may affect the cross talk between hematopoiesis and the microbiota, potentially exacerbating inflammatory or infectious states in the host. The Journal of Immunology, 2014, 193: 5273–5283.

5274 culture of BM (12). Decreased numbers of granulocytes have also been observed in the BM of kanamycin-treated mice (13). More recent studies have identified increased bone mass and decreased osteoclast numbers in GF mice (14). Thus, we hypothesized that cross talk between the microbiota and the BM is important to regulate the size of the steady-state myeloid cell pool. This regulatory network may be implicated in the increased susceptibility to infections observed in GF mice and patients with dysbiosis.

Materials and Methods Animal experiments

Monocolonizations, triple colonizations, antibiotic treatment, and i.v. injections Stable Escherichia coli K-12 JM83 monocolonizations, Enterococcus faecalis (mouse isolate) monocolonizations, and triple colonizations with E. coli K-12 JM83, S. xylosus (mouse isolate), and Enterococcus faecalis were done by intragastric gavage of GF animals with 109 CFUs of purecultured bacteria in 500 ml. Inocula were aseptically prepared and imported into flexible film isolators. This mixture of bacteria was chosen based on our previously published work demonstrating absence of pathology in MyD882/2TICAM12/2 animals (9), ease of culture and quantification, and to examine two relevant isolates commonly found in SPF mouse colonies alongside an apathogenic Gammaproteobacteria strain. Antibiotic-treated mice were administered 1 mg/ml metronidazole, 1 mg/ml ampicillin, 0.5 mg/ml neomycin sulfate, and 1 mg/ml vancomycin in the drinking water (refreshed every 4 d) for 4 wk before analysis. GF, E. faecalis monocolonized, and triple-colonized mice received 107 live E. coli K-12 i.v. into the tail vein. Blood was collected retro-orbitally using sterile glass Pasteur pipettes under isoflurane anesthesia (Halocarbon Laboratories) 30 min, 3 h, or 6 h after injection of live bacteria. Animals were euthanized and spleen, liver, lungs, and BM were removed aseptically. Fecal pellets were collected, the cecum was opened, and an aliquot of cecal content was taken. Organs were homogenized in 0.5% Tergitol/PBS using a Tissuelyser (Qiagen) and sterile stainless-steel ball bearings. Cecal contents, blood, and organ suspensions were then plated on Luria–Bertani (LB) containing appropriate antibiotics for overnight culture at 37˚C and CFU counting.

i.v. injection of heat-killed bacteria and serum Eight-week-old GF C57BL/6 mice were exported into sterile IVCs and injected into the tail vein with heat-killed (20 min at 121˚C) E. coli 104 or 106 diluted in PBS to a final volume of 500 ml. Pooled serum from SPF or GF JH2/2 or RAG2/2 mice was sterile filtered and 400 ml injected per mouse. PBS was autoclaved, sterile filtered, and 500 ml injected per mouse. All mice were analyzed after 24 h, and GF status was confirmed by plating of feces on blood-agar plates and anaerobic incubation for 7 d at 37˚C, as well as liquid cultures in thioglycollate medium (#CM0173; Oxoid) for 2 wk.

Reversible HA107 colonization and microbiology Reversible colonization was performed as described previously (16). In brief, D-Ala (200 mg/ml)/m-diaminopimelic acid (50 mg/ml)–supplemented LB cultures were aseptically inoculated from single colonies of E. coli HA107 and incubated with shaking at 160 rpm at 37˚C for 18 h. Bacteria were harvested by centrifugation (15 min, 3500 3 g, 4˚C) in a sterile aerosol-proof assembly, washed in sterile PBS, and concentrated to a density of 2 3 1010 CFU/ml in PBS, all performed aseptically under a sterile laminar flow hood. The bacterial suspensions were sealed in sterile

tubes, with the outside surface kept sterile, and imported into flexible film isolators, where 500 ml (1010 CFU) was gavaged into the stomachs of GF C57BL/6 mice. Inoculum samples were then re-exported from the isolators for bacterial quantification by plating on supplemented agar plates. Fecal samples exported from the isolator were bacteriologically analyzed to monitor HA107 shedding and bacteriological status of the inoculated mice.

BrdU proliferation assay GF or separately housed SPF C57BL/6 mice were i.p. injected with 1 mg BrdU (BD Biosciences) 36 and 24 h before analysis and retro-orbitally bled 8, 24, or 72 h after the second injection into sterile EDTA tubes. Femurs and tibias were collected and BM recovered by flushing in 2 ml RPMI 1640. Cells were surface-stained using Pacific blue–anti-Ly6G (Biolegend), PerCP–Cy5.5–anti-Ly6C (eBioscience), allophycocyanin–Cy7–anti-CD11b (Biolegend), and FITC–anti-B220 (Biolegend) Abs in 50 ml PBS/2% BSA, fixed, permeabilized using Cytofix/Cytoperm (BD Biosciences) and Cytofix/ Cytoperm Plus (BD Biosciences) solutions, and intracellularly stained with PE-conjugated anti-BrdU Ab or PE-isotype control (BD Biosciences) following the manufacturer’s instructions. Samples were then acquired on a BD LSR II flow cytometer, and data were analyzed using FlowJo Software (Tree Star).

FACS analysis Fifty microliters whole blood or BM were incubated with 50 ml PBS/ BSA2% containing Pacific blue–anti-Ly6G (Biolegend), PerCP–Cy5.5– anti-Ly6C (eBioscience), allophycocyanin–Cy7–anti-CD11b (Biolegend), FITC–anti-B220 (Biolegend), and PE–anti-CD54 (Biolegend) Abs, incubated 20 min on ice, and RBC lysis was performed using FACSLysis solution (BD Biosciences). Cells were washed in PBS/BSA 2%, and all samples were acquired on a BD LSR II Flow Cytometer. Data analysis was performed using FlowJo Software (Tree Star), and frequencies were normalized to the total number of leukocytes as determined using a VetABC animal blood counter (Medical Solution) or a hemocytometer (BM). Myeloid cells were identified by expression of CD11b and absence of B220, and then further subdivided into Ly6G+ granulocytes and Ly6C+ monocytes.

Bacterial FACS analysis Bacterial FACS analysis was performed as described previously (11, 17). In brief, single colonies of plated bacteria were inoculated into 5 ml LB medium and cultured overnight at 37˚C. Bacteria were then gently pelleted for 3 min at 7000 rpm in an Eppendorf minifuge and washed three times with sterile-filtered PBS/2% BSA/azide before determining the OD600 and resuspending at ∼107 bacteria/ml. Mouse serum was diluted 1:10 in PBS/ 2% BSA/azide and heat-inactivated at 60˚C for 30 min. The serum solution was then spun at 13,000 rpm in an Eppendorf minifuge for 10 min to remove any bacteria-sized contaminants, and the supernatant was used to perform serial dilutions. Serum solution and bacterial suspension were then mixed at a ratio of 1:1 and incubated at 4˚C for 1 h. Bacteria were washed twice before resuspending in monoclonal PE anti-mouse IgG1 and allophycocyanin anti-mouse IgM (BD Pharmingen). After a further hour of incubation, the bacteria were washed and then resuspended in 2% paraformaldehyde/PBS for acquisition by FACSArray using forward scatter and side scatter parameters in logarithmic mode.

Progenitor cell analysis Femurs and tibias were flushed into 20 ml RPMI 1640, and whole BM cells were counted in a hemocytometer using Trypan blue staining to exclude dead cells. BM lineage depletion was performed using biotinylated Abs against red cell precursors (anti-Ter119), B cells (anti-CD19, clone 6D5), T cells (anti-CD3ε, clone 145-2C11), and myeloid cells (anti-Gr1, clone RB6-8C5; all Biolegend), MACS anti-biotin beads, and LS columns (Miltenyi Biotec). Cells were then stained with anti-CD127–FITC (SB/ 199), anti-CD135–PE (A2F10), anti-CD16/32–PE–Cy7, anti-Thy1.1– allophycocyanin (OX-7), anti–c-kit–allophycocyanin–Alexa750 (all Biolegend); anti–Sca-1–PerCP–Cy5.5 (D7) and anti-CD34–eFluor 450 (RAM34; eBioscience). The hematopoietic stem/progenitor cell (HSPC)–enriched fraction was identified by the lack of lineage markers (lin2) and by the expression of Sca-1 and c-kit (lineage2, Sca1+, c-kit “high” cells [LSK]). Granulocyte-monocyte progenitors (GMPs) were identified from the myeloid lineage-restricted progenitors (c-kit+, Sca-12, CD1272) according to FcgR and CD34 expression.

Total BM CFU assay For Figs. 2 and 3, total BM cells (3.0 3 103 cells/well) were cultured for 12–14 d in methylcellulose media (MethoCult M3234; Stemcell Technologies) supplemented with mouse IL-3 (10 ng/ml), human IL-6 (10 ng/ml),

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C57BL/6, MyD882/2TICAM12/2, and NMRI mice were rederived GF as previously described (9, 11), and maintained GF in flexible film isolators at the Clean Animal Facility, University of Bern, Switzerland. C57BL/6 mouse colonies associated with a low-complexity microbiota (LCM) consisting of 10–100 different bacterial species (15) were originally generated by selective colonization of GF mice. Conventionally raised laboratory SPF mice were housed in individually ventilated cages (IVCs) at the Central Animal Facilities, University of Bern. All animals were housed in Bern, Switzerland, except for LCM mice shown in Figs. 2 and 4, which were maintained in a full-barrier facility in IVC cages at the Swiss Federal Institute of Technology (ETH) Zurich. In all experiments, mice were fed the standard mouse chow Kilba Nafag 3437.P.M.L15 M/R autoclaved at either 121˚C (SPF and LCM mice in IVC housing) or 132˚C (GF and gnotobiotic mice). All animal experiments were approved by the local Animal Care Committee.

THE MICROBIOTA DRIVES STEADY-STATE GRANULOPOIESIS

The Journal of Immunology mouse stem cell factor (10 ng/ml), mouse GM-CSF (10 ng/ml), mouse thrombopoietin (10 ng/ml), and human erythropoietin (10 U/ml; all Peprotech). The colonies were counted on the basis of their morphological characteristics (18).

Lin2 CFU assay For Fig. 4, a total of 3.3 3 103 MACS-purified lin2 BM cells were plated into MethoCult M3134 medium (Stemcell Technologies) supplemented with 15% FCS, 20% BIT (50 mg/ml BSA in IMDM, 1.44 U/ ml human insulin [Actrapid; Novo Nordisk], and 250 ng/ml human holo transferrin [Prospec]), 100 mM 2-ME, 100 U/ml penicillin, 100 mg/ml streptomycin, 2 mM L-glutamine, and 50 ng/ml mouse SCF, 10 ng/ml mouse IL-3, 10 ng/ml human IL-6, and 50 ng/ml mouse Flt3-ligand (all from Prospec). Colonies were enumerated after 7 d ($30 cells/colony) on a DMIL inverted microscope (Leica) equipped with an Intensilight C-HGFI unit (Nikon) (17).

Serum lipocalin 2 ELISA

Serum cytokine measurements Serum cytokines were measured using cytometric bead arrays (BD Biosciences). Serum was 1:4 diluted and incubated for 1 h with 0.5 ml capture beads per sample (Mouse IL-6 flex set B4 #558301, Mouse TNF flex set C8 #558299, Mouse MCP-1 flex set B7 #558342; BD Biosciences). After washing in PBS, 0.5 ml PE-detection beads was added per sample and incubated 1 h at room temperature. Samples were then washed in PBS and acquired on a FACS Array (BD Biosciences). Four-parameter standard curves were fitted for each cytokine standard, and concentrations of cytokines in the serum samples were calculated.

Serum LPS measurements Serum LPS concentrations were determined using the Limulus Assay kit according to the manufacturer’s protocol (50-647U, 50-648U; Lonza).

Statistical analysis Differences were analyzed for statistical significance using Prism 4 for Macintosh (GraphPad Software). The details of the test carried out are indicated in the figure legends. Where data were approximately normally distributed, values were compared using either a Student t test for single variable or two-way ANOVA for two variables. Approximate p values were computed for two-way ANOVA. Where data were not normally distributed (e.g., bacterial CFU counts close to or equal to zero), nonparametric two-tailed Mann–Whitney U tests were applied. In all cases, p , 0.05 was considered significant.

Results We and others have previously reported that GF mice show impaired clearance of systemically administered bacteria, even when the bacterial strain given is fully avirulent (9, 10). To confirm these observations, we compared clearance of an i.v. dose of ampicillinresistant E. coli K-12 from the blood of GF C57BL/6 mice, with clearance from littermates that had been colonized for 21 d with E. faecalis, S. xylosus, and ampicillin-susceptible E. coli K-12. At 6 h after injection, GF mice had higher counts of ampicillin-resistant E. coli K-12 in their blood (Fig. 1A). Because E. coli K-12 was present in the intestines of the colonized mice, we first sought to exclude a role for Ag-specific adaptive immunity in the improved bacterial clearance. Although intestinal secretory IgA specific for

E. coli K-12 had been induced in the colonized animals (data not shown), this isotype at mucosal surfaces is not thought to augment bacterial clearance from the peripheral blood (19), and colonization of clean mice induces mucosal IgA independently of serum IgG induction (20). In the serum, there was no measurable specific IgG or IgM (Fig. 1B, 1C) directed against E. coli K-12 in either GF or colonized animals. As a positive control, we compared the titer of E. coli–specific IgG present in the serum of E. coli K-12 monocolonized MyD882/2TICAM12/2 mice, which are known to mount strong systemic Ab responses to their intestinal bacteria because of failed mucosal containment (9) (Fig. 1B, inset). Supporting the interpretation that the differences in innate immunity likely underlie the delay in systemic vascular bacterial clearance in the GF state, our previous data demonstrated an identical phenomenon when we compared i.v. E. coli K-12 challenge of GF wild-type mice and mice colonized with the evolutionarily distant Firmicute E. faecalis (9). No Ab cross-reactivity could be observed between these two bacterial strains (9). We also confirmed experimentally that innate immune responses were enhanced during peripheral blood bacteremia in mice carrying intestinal microbes. After i.v. challenge there were enhanced responses of serum lipocalin 2 (Fig. 1D) and the myeloid-associated cytokines and chemokines IL-6 (Fig. 1E), MCP1 (Fig. 1F), and TNF (Fig. 1G) in colonized compared with GF mice, despite equal steady-state levels of serum lipocalin 2 in unmanipulated animals, regardless of their colonization status (Fig. 1H). Initial clearance of bacteria from the blood involves both phagocytes and bactericidal serum factors such as the complement cascade. As murine complement activity is difficult to measure because of its labile nature (21) and complement factor production has previously been described to be normal in GF and Ag-free mice (22, 23), we decided to determine whether the microbiota was linked to steady-state myelopoiesis. We first quantified blood and BM HSPCs (which are found in the LSK fraction), granulocyte precursor populations, and mature granulocytes (Fig. 2A, 2B) from mice housed under four different hygiene conditions with increasing microbiota complexity: 1) GF, that is, mice housed stringently in isolators that have never encountered live microbes; 2) “triple-colonized mice,” which were generated by experimentally colonizing GF mice from pure cultures of E. coli K-12, E. faecalis, and S. xylosus; 3) LCM mice harboring between 20 and 100 bacterial strains but lacking any Gammaproteobacteria (4, 15) SPF mice, which were bred and maintained with a full microbiota of .100 bacterial species, but screened to be free of known mouse pathogens. In agreement with the literature on fully GF and antibiotic-treated mice (7, 12, 13), the number of myeloid cells, mature granulocytes, mature monocytes, and GMPs in BM all increased consistently with increasing microbiota complexity (Fig. 2C–F), suggesting the existence of a microbiota-dependent signal that increases myeloid cell production in healthy animals. The LSK cell population was only increased in the presence of a complete “SPF” microbiota, suggesting that amplification of this population requires either a stronger microbiota-associated signal or the presence of microbial species absent from gnotobiotic mice (Fig. 2G). The size of the common lymphoid progenitor (CLP) population did not differ significantly with hygiene status (Fig. 2H). These changes were also observed in classical CFU assays of total BM from GF and SPF mice, confirming a difference in the HSPC frequencies on a functional basis (Fig. 2I). In addition, SPF mice treated with broad-spectrum antibiotics showed a corresponding decrease in BM granulocyte and in the LSK cell numbers (Fig. 3A, 3B). The antibiotic-induced decrease in myelopoiesis could also be observed at the functional level in total BM CFU assays (Fig. 3C). Thus, the presence of an intestinal

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Lipocalin 2 ELISAs were performed according to the manufacturer’s instructions with a few modifications (murine; R&D Systems). Nunc ELISA plates were coated with 50 ml capture Ab (1:200 in PBS) overnight at 4˚C in a humidified chamber. After washing in PBS/Tween 0.05% (Sigma-Aldrich) and blocking in 150 ml PBS/BSA 2% for 15 min at room temperature, samples and standards were added in 3-fold dose titrations starting at 1:10 (serum and standard) and incubated overnight at 4˚C in a humidified chamber. After washing in PBS/Tween 0.05%, 50 ml detection Ab (1:200 in PBS/BSA 2%) was added and plates were incubated for 1 h at room temperature. Plates were then washed in PBS/Tween 0.05% and 100 ml HRP-streptavidin (1:1000 in PBS; Biolegend) was added for 1 h. Plates were then washed and developed with 100 ml of substrate solution (10 ml substrate buffer, 1 mg ABTS, 10 ml H2O2 [both from SigmaAldrich]). OD was measured at 415 nm and four-parameter curves were generated to compare EC50 values of samples and standards.

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THE MICROBIOTA DRIVES STEADY-STATE GRANULOPOIESIS

microbiota appeared to specifically amplify myelopoiesis in the BM, and with simple microbiota, this amplification occurred mainly between the production of multipotent progenitors and their differentiation into GMPs. We next examined whether changes in BM populations induced by the microbiota were dynamic or represented an irreversible developmental shift in response to colonization. Thus, GF mice were treated with the strongly auxotrophic E. coli K-12 mutant HA107 (16). This bacterium colonizes the intestine for 12–24 h postgavage but cannot replicate in vivo, so GF status is regained between 24 and 48 h postgavage (16). At 12 h postgavage, an expansion of the BM myeloid cell pool was observed (Fig. 4A, 4B). However, 14 d postgavage, that is, 13 d after regaining GF status, HA107-treated mice were equivalent to GF mice in their BM myeloid cell status (Fig. 4A, 4B). Although the changes in myeloid progenitor cells did not reach significance in these experiments (Fig. 4C), we could observe an increase in CFU formation 12 h after HA107 treatment that returned to baseline 14 d after regaining GF status (Fig. 4D). Together, these data imply that changes in myeloid cell production are regulated dynamically depending on the current level of microbiota exposure. In contrast with the BM, no significant differences were observed in peripheral blood granulocyte or monocyte numbers between any of the colonization statuses (Fig. 5A–C). We therefore used a 24-h pulse of BrdU labeling to address the kinetics of granulocyte production and release in GF and SPF mice. Although the total number of HSPCs, GMPs, and granulocytes in BM was consistently higher in SPF mice, the fractions of these populations incorporating label over time was identical between GF and SPF animals (Fig. 6A, 6B). This is consistent with published observations (7) and may indicate that different population levels are

established early after colonization, but no steady-state kinetic differences in myelopoiesis between GF and SPF mice. However, the fraction of BrdU+ cells within any FACS marker–defined cell population is a complex function of influx from precursor populations, the rate of cell division within the defined population, the rate of death within the population, and the rate of differentiation into subsequent populations (24): any of these components may differ in GF and colonized animals. Alternatively, because the absolute numerical difference between GMPs in GF and SPF BM was ∼2-fold, either transient differences at the time of initial colonization or very small steady-state differences in the proliferation, immigration, and emigration rates that are not detectable within the resolution of our BrdU analysis could also sufficiently explain the observed data. Blood granulocytes are terminally differentiated cells that are thought to be incapable of further cell division (postmitotic) and are directly derived from the nonreplicating BM granulocyte pool. Thus, the fraction of BrdU+ granulocytes in blood depends only on the rate of cell influx from the BM pool and the rate of granulocyte removal by death or tissue emigration. Because the steady-state number of granulocytes in blood is similar in GF and SPF mice (Fig. 5B), these two rates must be approximately equal. Thus, the significantly decreased fraction of BrdU+ blood granulocytes that is measured 48 h postlabeling in GF mice (Fig. 6C) represents a decreased rate of influx of granulocytes into the blood and a correspondingly decreased rate of clearance of granulocytes from the blood in GF mice. Extending this logic, the rate of influx into the blood granulocyte population must be equal to the rate of efflux from the BM granulocyte population. However, no difference in the labeling kinetics between GF and SPF BM granulocytes was observed

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FIGURE 1. GF mice show delayed clearance of apathogenic bacteria and blunted inflammatory responses. (A) GF C57BL/6 mice 10 wk of age were triple-colonized by oral gavage of 107 CFU each of E. coli K-12, S. xylosus, and E. faecalis. After 21 d of colonization, triple-colonized mice and GF controls were then i.v. injected with 107 CFU E. coli K-12, and bacterial counts were determined 6 h later in the peripheral blood. Each point represents one mouse from one of two independent experiments, and horizontal lines show geometric means. (B and C) Serum IgG and IgM titers against E. coli K-12 of the mice shown in (A). Dose titrations of serum were incubated with E. coli K-12, and specific IgG- and IgM-binding were visualized by FACS. All curves represent individual mice from one of two independent experiments. (B, inset) Serum IgG against E. coli K-12 from wild-type (open symbols) and Myd882/2 TICAM12/2 mice monocolonized with E. coli K-12. (D) Serum lipocalin 2 values 6 h after i.v. injection of 107 CFU E. coli K-12 in the same mice as in (A) and (B). Each point represents one mouse and horizontal lines show geometric means. (E–G) Serum cytokine levels 30 min, 3 h, and 6 h after i.v. injection of 107 CFU E. coli K-12 in GF (open circles and dashed lines) or triple-colonized (filled circles and continuous lines) mice. Each point represents one mouse from one of two independent experiments. (H) Serum lipocalin 2 values in unmanipulated GF and triple-colonized mice. Each point represents one mouse and horizontal lines show geometric means. Unpaired t test was used to compare GF and triple-colonized mice, *p # 0.05, **p # 0.01.

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FIGURE 2. Size of myeloid pool corresponds to microbiota complexity. (A and B) Gating strategies used in (C)–(H). The HSPC-enriched fraction was defined as LSK. GMPs were identified from the myeloid lineage-restricted progenitors (c-kit+, Sca-12, CD1272) according to FcgR and CD34 expression. Myeloid cells were identified by expression of CD11b and absence of B220, and then further subdivided into Ly6G+ granulocytes and Ly6C+ monocytes. (C–H) Absolute numbers of myeloid cells, granulocytes, monocytes, GMP, LSK, and CLP in the BM of C57BL/6 mice 7–9 wk of age kept GF, colonized for 21 d with E. coli, S. xylosus, and E. faecalis by oral gavage (triple), harboring an LCM or kept under SPF conditions. Each point represents an individual mouse pooled from one to four independent experiments, and horizontal lines show means. One-way ANOVA and Tukey’s posttest were used to compare the groups. *p # 0.05, **p # 0.01, ***p # 0.001. (I) CFU numbers per hind leg (femur + tibia) of C57BL/6 mice kept under GF or SPF conditions. Student t test was performed to compare total or each type of colonies between the groups. *p , 0.05 (for total CFU and CFU-M). BFU-E, burst forming uniterythrocyte; CFU-G, CFU-granulocyte; CFU-GEMM, CFU-granulocyte/erythrocyte/macrophage/megakaryocyte; CFU-GM, CFU-granulocyte/macrophage; CFU-M, CFU-macrophage; CFU-Mk, CFU-megakaryocyte.

(Fig. 6D). This may be because of matched decreased differentiation and proliferation rates throughout the whole myeloid lineage, or may simply be too small an effect to see above biological noise, as only 1–2% of the total granulocyte population is in the circulation at any one time (25).

In summary, in GF mice, the BM granulocyte pool and myeloid precursors are present at half to one quarter of the normal numbers observed in SPF animals. This corresponds to slower kinetics of granulocyte turnover in the peripheral blood. Using our current methods, no change in the kinetics of BM myelopoiesis could be

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FIGURE 3. Treatment with broad-spectrum antibiotics decreases myelopoiesis. (A and B) Absolute numbers of granulocytes (A) and LSK cells (B) in the BM of C57BL/6 mice maintained with antibiotics (metronidazole, neomycin, ampicillin, and vancomycin: Abx) or without antibiotics (control) in drinking water. Each point represents an individual mouse pooled from three independent experiments, and horizontal lines show means. Student t test was used to compare the groups. (C) CFU numbers per hind leg of C57BL/6 mice kept for 4 wk with antibiotics (Abx) or without antibiotics (control) in drinking water. Data show means + SEM and are pooled from three independent experiments with a total of three or five mice (control or Abx, respectively) per group. Student t test was performed to compare total or each type of colony between the groups. *p , 0.05, ***p , 0.001. BFU-E, burst-forming unit-erythrocyte; CFU-G, CFU-granulocyte; CFU-GEMM, CFU-granulocyte/erythrocyte/macrophage/megakaryocyte; CFU-GM, CFU-granulocyte/macrophage; CFU-M, CFU-macrophage; CFU-Mk, CFU-megakaryocyte.

FIGURE 4. Microbiota-driven myelopoiesis is dynamically regulated. Absolute numbers of BM myeloid cells (A), granulocytes (B), GMPs (C), and lin2 CFU (D) in 6- to 8-wk-old C57BL/6 mice. GF mice were gavaged four times over 2 wk with 1010 E. coli HA107 and analyzed 12 h after the last gavage, when the intestine was still colonized (“HA107 12h transiently colonized,” filled squares), or 14 d later, when the mice were GF again (“HA107 d14 GF,” open squares) in comparison with GF mice (open circles). Pooled data from two independent experiments are shown. Each point represents an individual mouse, and horizontal lines show means. One-way ANOVA and Tukey’s posttest were used to compare the groups. *p # 0.05.

phenomenon could be observed already at 24 h after serum transfer, indicating a very rapid effect on the myelopoietic lineage. Correspondingly, these treatments were also associated with an increase in the fraction of immature granulocytes in the blood, as revealed by a mild “left shift” of the granulocyte population (18) (Fig. 7C, 7D). The heat stability of the active serum compound driving myelopoiesis suggested that microbial products in the serum might be necessary to maintain a sufficient myeloid cell pool. This was a surprising suggestion given the known firewall effect of the mesenteric lymph nodes and liver in preventing systemic spread of live microbes (26, 27), and the absence of microbiota-specific systemic adaptive immunity in healthy mice (9, 28). We therefore hypothesized that only concentrations of microbial products well below the threshold required to activate systemic adaptive immunity would be sufficient to recapitulate the myeloid cell expansion observed on colonization. Wild-type GF mice received injections of either 104 or 106 heat-killed E. coli i.v. to test this.

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observed between GF and SPF animals. Taken together with the rapid dynamic responses seen during reversible colonization with HA107, this suggests that the increased output in steady-state is rather related to expansion of the BM that has occurred soon after colonization (shortly after birth) in SPF mice. To define how the intestinal microbiota might be communicating with the BM, we attempted to replicate the colonization phenotype by sterile transfer of serum from SPF mice or GF mice into otherwise unmanipulated GF mice. This transfer of SPF but not GF serum was sufficient to greatly expand the BM myeloid cell pools (Fig. 7A, 7B) without affecting the peripheral blood granulocyte numbers (data not shown). Intriguingly, transfer of an equivalent volume of serum in which the majority of protein had been fully denatured (data not shown) and precipitated by boiling at 90˚C for 30 min, followed by high-speed centrifugation and sterile filtration, showed an enhanced effect on BM myelopoiesis when compared with untreated serum, demonstrating that the active component is highly heat stable (Fig. 7A, 7B). Of interest, this

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FIGURE 5. Peripheral blood myeloid cell numbers are similar with different levels of microbiota complexity. (A–C) Absolute numbers of peripheral blood myeloid cells, granulocytes, and monocytes in C57BL/6 mice at 7–9 wk of age, kept GF, colonized for 21 d with E. coli, S. xylosus, and E. faecalis by oral gavage (triple), LCM, and SPF. Each point represents one individual mouse pooled from one to four independent experiments, and horizontal lines show means. Myeloid cells were identified by expression of CD11b and absence of B220, and then further subdivided into Ly6G+ granulocytes and Ly6C+ monocytes as shown in Fig. 2B.

FIGURE 6. BM BrdU labeling follows similar kinetics in GF and SPF mice, but labeled granulocytes accumulate faster in the peripheral blood of SPF mice. (A–D) Time course of appearance of labeled LSK, GMP, and granulocytes in BM (A, B, and D) or peripheral blood (C) after two i.p. injections of 1 mg BrdU in 7-wk-old C57BL/6 GF (open circles and dashed lines) or SPF (filled dots and continuous lines) mice. Data show means 6 SD from two independent experiments with n = 3 mice per group. Unpaired t test was used to compare the groups. *p # 0.05. Myeloid cells were identified by expression of CD11b and absence of B220, and then further subdivided into Ly6G+ granulocytes and Ly6C+ monocytes (as in Fig. 2B). The HSPC-enriched fraction was defined as lin2, Sca-1+, c-kithi (LSK). GMPs were identified from the myeloid lineage-restricted progenitors (c-kit+, Sca-12, CD1272) according to FcgR and CD34 expression (as in Fig. 2A).

required for signaling via IL-1 family cytokine receptors, but because these cytokines should be destroyed by the vigorous heatinactivation process used and were undetectable in the heat-treated sera (data not shown), these data strongly suggested that heatstable microbial compounds present in SPF mouse serum are necessary to drive sufficient steady-state myelopoiesis. Correspondingly, MyD882/2TICAM12/2 mice monocolonized with E. faecalis did not display improved clearance of E. coli (Fig. 8F).

Discussion It is now well established that the innate immune system is required to maintain homeostasis with the microbiota (9, 31–33). However, the details of cross talk between the intestinal microbiota and the host are only starting to be realized. In this article, we reveal that systemic recognition of microbiota-derived products by TLRs is necessary to maintain a sufficient pool of BM myeloid cells. This suggests that coevolution of the host with its resident microbes has led to a reliance on microbiota-derived signals to maintain vigilance to infection. At first, it seems an odd evolutionary trajectory to have been selected, as surely it would be safer to genetically encode this level of myeloid cell production within the host genome. In contrast, changes in microbiota composition can be strong indicators of increased infectious challenge, for example,

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The lowest dose of heat-killed bacteria accurately recapitulated the effects of colonization 24 h postinjection, whereas higher doses resulted in complex phenotypes, most likely associated with high levels of cytokine production and rapid release and consumption of BM granulocytes, as would be expected in sepsisassociated responses (1, 6, 29) (Fig. 7E, 7F). Strong candidates for recognition and signaling downstream of systemic microbial compounds are TLRs (30). Indeed, lower rates of emergency myelopoiesis have been previously reported in MyD882/2 animals (5, 18, 29), although continuously elevated systemic bacterial counts in SPF MyD882/2 mice (9) could be associated with exhaustion of the myeloid system, complicating interpretation of these results. To determine whether TLR signaling is necessary for microbiota-induced increases in the myeloid cell pool, we carried out “triple” recolonizations of GF MyD882/2 TICAM12/2 mice and examined BM myeloid cell populations 21 d postgavage. In agreement with observations in SPF mice, no amplification of the BM myeloid cell pool was observed in MyD882/2TICAM12/2 mice (Fig. 8A–C), despite elevated systemic bacterial exposure (9). In addition, transfer of sterile-filtered, boiled serum from wild-type SPF mice into GF MyD882/2TICAM12/2 mice failed to induce any expansion of the BM myeloid compartment (Fig. 8D, 8E). MyD88 is also

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FIGURE 7. Transfer of serum from SPF mice or low numbers of heat-killed E. coli can mimic the effects of colonization. (A and B) Absolute numbers of BM myeloid cells and granulocytes in 8-wk-old C57BL/6 GF mice before (GF) or 24 h after i.v. injection of 400 ml PBS or sterile-filtered serum (serumtransfer). Serum was obtained from Ab-deficient GF or SPF mice and transferred before (SPF) and after heat inactivation (HI-SPF) for 30 min at 90˚C. Each point represents one mouse, and horizontal lines show means. Data shown are pooled from two independent experiments. *p # 0.05. (C) Percentage of CD11b+, Ly6Glow immature granulocytes among all granulocytes in peripheral blood of the same mice as in (A) and (B). (D) Representative FACS plots of peripheral blood leukocytes, pregated on Ly6G+ and Ly6C2 cells as shown in Fig. 2, 24 h after serum transfer of GF (left panel) or heat-inactivated SPFserum (right panel). (E and F) Absolute numbers of BM myeloid cells and granulocytes in 8-wk-old GF NMRI mice 24 h after i.v. injection of heat-killed E. coli K-12 in two different concentrations (104 and 106) or PBS control (PBS). Each point represents an individual mouse from one experiment, and horizontal lines show means. One-way ANOVA was used to compare the groups. *p # 0.05.

overgrowth of Gammaproteobacteria can be associated with increased susceptibility to intestinal colonization with pathogens of the same phylum (15). It further seems likely that “microbiota complexity” alone is not sufficient to explain the stimulus for myeloid cell expansion. Microbial species display highly variable relationships to the host, both with respect to the niche that they colonize (adhesion to epithelial cells, mucus, luminal) and to the microbial products that they produce (34). It remains highly possible that particular species and their constituent molecular

products are much stronger stimuli to the systemic innate immune system than others, and this will be an important area for future systems-biochemistry research. In this study, animals were not perfused before analysis, and we therefore do not directly distinguish between “marginated” and “circulating” granulocytes in the BM. Nevertheless, because the concentration of granulocytes in peripheral blood does not differ significantly between GF and colonized animals, the increase in mature BM granulocytes would be likely indicative of an increase

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in the marginated pool of granulocytes, as well as an increased rate of release and consumption, as revealed by BrdU labeling. The importance of the marginated pool size is illustrated by human individuals with benign ethnic neutropenia, in whom expansion of this pool in the BM is thought to fully compensate the low circulating granulocyte numbers in the peripheral blood (35). Interestingly, although we saw no differences in the blood neutrophil numbers between GF and colonized mice, as the complexity of the microbiota increased, there was a 2- to 3-fold expansion of the total BM granulocyte population, which may represent the marginated pool, although this compartmentalization was not directly addressed. Steady-state granulopoiesis contrasts markedly with the situation in emergency myelopoiesis, where the marginated pool is rapidly mobilized into the circulation, reducing the total BM granulocyte numbers, at the same time as massively increasing the rate of granulocyte production at the expense of other hematopoietic lineages (5, 6). In keeping with the homeostatic nature of colonization-induced myelopoiesis, CLPs are unaffected by colonization. A further possible complication in interpreting the data is the existence of reverse migration of neutrophils to BM, either in steady-state (36) or as a clearance mechanism for aged cells (37). Nevertheless, the reverse migrating population, where observed, makes up ∼0.25% of circulating neutrophils in a healthy animal, so this phenomenon is unlikely to contribute dramatically to the doubling in the BM granulocyte pool observed upon colonization of GF mice. Within the scope of this study we cannot exclude the role of other mechanisms in controlling the size of the myeloid cell pool in GF and colonized animals. Indeed, the decreased blood granulocyte

turnover in GF mice (Fig. 6C) is indicative that there is a lower rate of granulocyte consumption in GF mice. Numerous previous studies have indicated that the diurnal pattern of granulocyte release and removal can strongly influence the rate of granulocyte production (38). However, Ab-induced neutropenia was shown to increase myelopoiesis identically in GF and SPF mice (7), suggesting that this mechanism is microbiota independent. Other studies have suggested that IL-17 (39, 40) and IFN-g (8) play important roles in steady-state granulopoiesis, and that the production of these cytokines is known to be lower in the intestines of GF mice than of SPF animals (41, 42). Additional molecules originating from the microbiota, such as bile acid metabolites or short-chain fatty acids, are also known to shape the host immune system, for example, in the differentiation of regulatory T cells (39) or the activation status of intestinal macrophages (43). Such components are likely to be present in SPF mouse serum and be absent from GF mouse serum, and may not be fully inactivated by heat treatment; thus, we cannot exclude their role in modulation of granulocyte production. However, the complete absence of response of MyD882/2TICAM12/2 mice to either recolonization or serum transfer suggests that TLR stimulation is both necessary and epistatic to any other regulatory mechanisms. Intriguingly, TLR signaling is also essential for induction of emergency hematopoiesis (6, 29), suggesting the existence of a quantitative, rather than a “black and white,” switch from the steady-state. It is currently unclear whether there are differences in TLR sensitivity of different cell types, or differential signaling induced by low and high concentrations of TLR ligands within the same cells. Because of technical and ethical limitations in making

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FIGURE 8. Microbiota-driven myelopoiesis requires MyD88/TICAM1 signaling. (A–C) Percentage of BM myeloid cells, granulocytes, and GMPs in 10wk-old GF and triple-colonized wild-type (B6) and MyD882/2TICAM12/2 mice (MyD88/TICAM1). Triple colonizations were performed by oral gavage of 107 CFU each of E. coli K-12, S. xylosus, E. faecalis, and animals analyzed after 21 d. Each point represents an individual mouse from one experiment, and horizontal lines show means. One-way ANOVA and Tukey’s posttest were used, *p # 0.05. (D and E) Absolute numbers of BM myeloid cells and granulocytes in 8-wk-old GF MyD882/2TICAM12/2 mice 24 h after i.v. injection of 400 ml sterile-filtered serum from Ab-deficient SPF mice or 400 ml of the same serum after 30 min of heat-inactivation at 90˚C. Each point represents an individual mouse from one experiment, and horizontal lines show means. (F) Bacterial counts in the spleens 6 h after i.v. injection of 107 CFU E. coli K-12 JM83 into 6- to 8-wk-old MyD882/2TICAM12/2 mice maintained GF or monocolonized with E. faecalis for 21 d. Each point represents an individual mouse from one experiment, and horizontal lines show means. Unpaired t test was used to compare the groups.

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Acknowledgments We thank J. Kirundi, A. Huguenin, and B. Flogerzi for technical support and C. Benarafa for helpful comments.

Disclosures The authors have no financial conflicts of interest.

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