nihms570341.pdf

Gut microbiota promotes hematopoiesis tocontrol bacterial

infection

Arya Khosravi

Alberto Yáñez

Jeremy G. Price

Andrew Chow

Miriam Merad

Helen S.

Goodridge

, and

Sarkis K. Mazmanian

1,*

Division of Biology and Biological Engineering, California Institute of Technology, Pasadena CA,

91125, USA

Regenerative Medicine Institute and Research Division of Immunology, Cedars-Sinai Medical

Center, Los Angeles CA 90048, USA

Department of Oncological Sciences, Tisch Cancer Institute and Immunology Institute, Mount

Sinai School of Medicine, New York, NY 10029, USA

Summary

The commensal microbiota impacts specific immune cell populations and their functions at

peripheral sites, such as gut mucosal tissues. However, it remains unknown whether gut

microbiota control immunity through regulation of hematopoiesis at primary immune sites. We

reveal that germ-free mice display reduced proportions and differentiation potential ofspecific

myeloid cellprogenitors of both yolk sac and bone marrow origin. Homeostaticinnate immune

defects may lead to impaired early responses to pathogens. Indeed, following systemic infection

with

Listeria monocytogenes

, germ-free and oral antibiotic treated mice display increased

pathogen burden and acute death. Re-colonization of germ-free mice with a complex microbiota

restores defects inmyelopoiesis and resistance to

Listeria

. Thesefindingsreveal that gut

bacteriadirect innate immune cell development via promoting hematopoiesis, contributing to our

appreciation of the deep evolutionary connection between mammals and their microbiota.

Introduction

The vast majority of our interactions with bacteriaare symbiotic in nature, consisting of

colonization by a complex and diverse microbiota that inhabit humans for life.Rather than

causing inflammation, commensal microbes largely direct beneficial immune functions and

Correspondence: sarkis@caltech.edu.

The authors declare no conflicts of interest related to this work.

Author notes

: A.K., M.M., H.S.G. and S.K.M. designed the research. A.K., A.Y., J.G.P. and A.C. carried out the experiments. A.K.,

M.M. and H.S.G. and S.K.M. wrote the manuscript.

Supplemental Information

: Supplemental Information includes four figures and can be found with this article online

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Author Manuscript

Cell Host Microbe

. Author manuscript; available in PMC 2015 March 12.

Published in final edited form as:

Cell Host Microbe

. 2014 March 12; 15(3): 374–381. doi:10.1016/j.chom.2014.02.006.

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often engender health. In particular, the microbiota shapesglobalimmune cell repertoires,

thereby altering host susceptibility to inflammation and infection at sites of colonization(Hill

et al., 2012; Kamada et al., 2012; Mazmanian et al., 2008; Naik et al., 2012). Specific gut

bacteria or bacterial products have been shown to suppressintestinal inflammation(e.g.,

colitis) in micethrough a variety of immune mechanisms (Atarashi et al., 2013; Round and

Mazmanian, 2010; Smith et al., 2013).Furthermore, the impact of commensal microbes on

host immune responses is not limited to mucosal interfaces, but extends tosystemic

compartments; gut microbes regulate immune responses that influence organ-specific

autoimmunity in animal models of multiple sclerosis, rheumatoid arthritis and type 1

diabetes(Lee et al., 2011; Markle et al., 2013; Wu et al., 2010). While numerous examples

illustrate how the microbiota contributes to immune function at mucosal and systemic sites,

little is known about the influencesofgut bacteria on cellular development within primary

immune tissues.

The immune system begins to develop

in utero

, but full maturation requires both genetic and

environmental signals that further shape immunity after birth. Lymphoid and myeloid cells

develop largely from hematopoietic stem cells (HSCs) within primary tissues, where

molecular cues orchestrate immune cell differentiation from uncommitted HSCs and

progenitor cells via regulation of transcription factors and epigenetic modifications

(Weissman, 1994). Additionally, certain phagocyte populations (including Langerhans cells

and microglia), derived from embryonic precursors, are maintained independently of

HSCs(Sieweke and Allen, 2013). Genetic contributions (i.e., molecular cues encoded by the

host genome) to lineage commitment pathways that control the myeloid repertoire are well

studied (Georgopoulos, 2002). However, environmental factors that influence hematopoiesis

have not been extensively defined. Based on emerging data that the microbiota represents an

integral environmental factor in shaping numerous features of the immune system, we

reasoned that gut bacteria may be controlling central immunity. We report herein that

commensal microbes promote the maintenance of both HSC and embryonic-derived

myeloid cells during steady-state conditions. The absence of commensal microbes leads to

defects in severalinnate immune cell populations (including neutrophils, monocytes and

macrophages) within systemic sites. By controlling the differentiation of innate immunity,

the gut microbiota prepares the host to rapidly mount immune responses upon pathogen

encounter, as germ-free and antibiotic treated mice are impaired in clearance of systemic

bacterial infection. Our study reveals that gut microbesevolved to actively shape immunity

at its core—via regulation of hematopoiesis.

Results

Germ-Free Animals Display Global Defects in Innate Immune Cells

The commensal gut microbiota profoundly influencescellular proportions, migration and

functions of various immune cell subsets. Recent studies have provided numerous examples

illustrating how gut bacteria modulate innate and adaptive immune responses at mucosal

surfaces during infection, inflammation and autoimmunity(Kamada et al., 2013; Round and

Mazmanian, 2009). With such pervasive effects, we reasoned that the microbiota may

regulate hematopoiesis—the developmental programming of the immune system. Initially,

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to determine if the microbiota has global effects on systemic immune cell populations, we

profiled myeloid cells in the spleen of colonized (SPF; specific pathogen free) and germ-free

(GF) mice. Indeed, GF animals display reduced proportions and total numbers of F4/80

and F4/80

cells compared to SPF mice (Figures 1A-C).F4/80

cells are mainly

macrophages, while F4/80

splenocytes are a heterogeneous population of macrophages,

monocytes and neutrophils (Schulz et al., 2012). Intriguingly, all three cell subsets are

reduced in GF mice(Figure S1A). Furthermore, treatment of SPF mice with antibiotics also

results in diminished myeloid cell populations in the spleen (Figure S1B). Thus, gut bacteria

dynamically influence innate immune cell proportions at secondary immune sites in the

periphery.

Myeloid cell precursors differentiate into various phagocyte lineages that are stored in the

bone marrow, which are a major source of cells that populate peripheral tissues(Geissmann

et al., 2010). The reduction of splenic macrophages, monocytes and neutrophils in GF mice

suggests that defects in host immunity may include compromiseddevelopment in primary

immune sites. Accordingly, we observed a reduction of myeloid cells within the bone

marrow of GF mice (Figures 1D-F). A similar decrease was observed in the liver, a site of

alternative immune cell development (Figure S1C). A global defect in myeloid cell

populations in primary immune sites of GF mice demonstrates that gut bacteria shape the

architecture of the immune system early in cellular development.

Commensal Microbes Enhance Myelopoiesis

We reasoned that reductions in several phagocytic cell subsets in GF mice may reflect a

primary defect in the maintenance of myeloid cell populations. To test if commensal

microbes promote myelopoiesis, we pulsed SPF and GF mice with 5-Ethynyl-2

′

deoxyuridine (EdU), a thymidine analog, to compare the percentage of dividing leukocytes.

Both F4/80

and F4/80

phagocytes from GF mice showed reduced EdU incorporation

compared to SPF animals (Figure 2A,B). F4/80

macrophages are largely derived from

embryonic yolk sac progenitors and are maintained independently of HSCs (Schulz et al.,

2012; Sieweke and Allen, 2013). F4/80

leukocytes, however, are of hematopoietic origin

and reduced EdU incorporation by these cells in GF mice indicates defects in the expansion

and/or differentiation of bone marrow progenitor cells (Schulz et al., 2012). These studies

uncover a role for commensal microbes in promoting the maintenance of both splenic yolk

sac-derived and HSC-derived myeloid cells.

The reduction of F4/80

cells in GF mice led us to further investigate the contribution of

commensal microbes on HSCs and myeloid progenitor cells in the bone marrow. No

differences were detected in the proportion or differentiation potential of LKS

cells (HSCs

and multipotent progenitors; MPPs), LKS

cells (total lineage-restricted progenitors), or

common myeloid progenitor cells (CMPs) between SPF and GF mice (Figure S2A-F).

Remarkably, GF miceare significant reduced in the proportion of bone marrow granulocyte

and/or monocyte progenitors (GMPs), identified as LKS

CD34

cells (Figure 2C).

GMPs consist of progenitor cells, downstream of HSCs and CMPs during hematopoiesis,

with restricted myeloid differentiation potential (Akashi et al., 2000). To further examine the

effects of gut microbiota on innate immune cells, we tested if commensal microbes affect

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the differentiation potential and self-maintenance capacity of GMPs. Methylcellulose culture

of LKS

CD34

cells from GF mice displayed reduced granulocyte (G-CFU) and

monocyte (M-CFU) colony formation compared to cells from SPF mice (Figure 2D).

Furthermore, passage of LKS

CD34

cells isolated from GF mice in primary

methylcellulose culture yielded reduced recovery of c-Kit

CD11b

progenitor cells

compared to SPF GMPs (Figure 2E). This suggeststhe ability of GMPs to maintain cells

with progenitor potential isdefective in the absence of commensal microbes(Rodrigues et al.,

2008). Consistent with this notion, secondary cultures of unfractionated cells derived from

GF GMPs generated fewer colonies compared to cells isolated from SPF mice (Figure 2F).

The commensal microbiota therefore promotes steady-state myelopoiesis by specifically

maintaining GMP proportions and enhancing their differentiation to mature myeloid cells in

the bone marrow.

Extramedullary hematopoiesis (outside the bone marrow) further contributes to the

maintenance and inflammatory responses of tissue-resident phagocytic cells (Jenkins et al.,

2011; Massberg et al., 2007; Robbins et al., 2012; Swirski et al., 2009). We therefore

investigated whether commensal microbes influence the hematopoietic potential of

progenitors located in the spleen. Similar to GMPs from the bone marrow, splenocytes

isolated from GF mice displayed reduced colony formation in methylcellulose, compared to

SPF mice, with significant reductions in both neutrophil and monocyte production (Figures

2G, H). Overall, we conclude that the microbiota shapes innate immune profiles by

promoting myeloid progenitor development and differentiation in the bone marrow and

extramedullary sites, revealing that gut bacteria control immunity at its core—during

hematopoiesis.

Tissue-Resident Phagocytes Mediate Protection by Commensal Microbes

Innate immune cells are the first responders to infection, mediating early pathogen control

and coordinating downstream immune reactions(Kastenmuller et al., 2012; Shi and Pamer,

2011). We sought to test the impact of commensal microbes on myeloid cell differentiation

by employing infection models where innate immunity is vital for an effective immune

response. SPF and GF mice were infected intravenously (

i.v.

) with the model pathogen,

Listeria monocytogenes

. SPF mice challenged systemically with

L. monocytogenes

effectively control infection, as previously described (Figure 3A) (Serbina et al., 2012; Shi

et al., 2011). However, GF mice rapidly succumb at the same inoculum (Figure 3A).

Heightened susceptibility to infection among GF mice was associated with a significant

increase in splenic and liver bacterial burden 24 and 72 hours post-infection (hpi),

demonstrating a defect in early resistance to

Listeria

infection (Figures 3B, C and Figure

S3A). Susceptibility to infection is not restricted to

L. monocytogenes

, as GF mice also

displayed increased disease burden following systemic challenge with

Staphylococcus

aureus

(Figure S3B). Interestingly, SPF mice treated orally with broad-spectrum antibiotics

were also impaired in controlling

Listeria

, indicating protection by commensal microbes is

an active process and is subject to loss following depletion of gut microbiota (Figure 3D).

Collectively, these data reveal that commensal microbes are critical for rapid and potent

systemic immune responses to acute bacterial infection.

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To confirm that defects in myelopoiesis contribute to increased disease burden in GF mice,

phagocytic cells were depleted with clodronate-loaded liposomes (CL) prior to infection

with

L. monocytogenes

(van Rooijen et al., 1996). CL pre-treatment increased susceptibility

Listeria

infection (Figure 3E), confirming the importance of resident cells in pathogen

resistance(Aichele et al., 2003; Kastenmuller et al., 2012). Importantly, depletion of resident

phagocytes rendered both SPF and GF mice equally susceptible to infection, resulting in

similar splenic disease burden 24 hpi (Figure 3E), and rapid death within 48 hpi (Figure 3F).

While functional defects inmyeloid cellsmay potentially contribute to increased disease in

GF mice, we did not detect differences during

in vitroListeria

killing by macrophages from

SPF or GF mice (Figure S3C). Furthermore, CD11b+ myeloid cells isolated from either SPF

or GF donors were equally sufficient in providing protection when transferred into GF mice

prior to infection (Figure 3G),suggesting reduced cell proportions are likely the primary

defect in GF mice. These studies confirm the importance of microbiota-driven myelopoiesis

in promoting host resistance during systemic infection.

Effective responses to

L. monocytogenes

requires coordination between innate and adaptive

immune cells, resulting in pathogen clearance and protective immunity (Pamer, 2004). Thus,

we investigated whether additional immune cells beyond tissue-resident phagocytes may

mediate commensal-derived protection to

Listeria

infection. We show that adaptive

immunity is not required for protection by the microbiota during acute infection (Figure

S3D), nor are GF mice deficient in developing long-term protective immunity against

subsequent infection (Figure S3E). Furthermore, the selective expansion of myeloid cells

during acute infection, called emergency hematopoiesis, which is necessary for mediating

delayed resistance to

L. monocytogenes

(following 48 hpi), was maintained in GF mice

(Figure S3F). Finally, while there are fewer inflammatory neutrophils and monocytes

recruited to the spleen following infection (Figure S3G), a possible consequence of

increased apoptosis (Figure S3H), these cells were not required for commensal-mediated

protection against

L. monocytogenes

(Figure S3I, J). Together, these findings demonstrate

that hematopoietic defects in tissue-resident myeloid cells prior to infection of GF mice (i.e.,

during cell development) is the primary cause of impaired control of

Listeria

CommensalBacterialSignals Mediate Maintenance of Myelopoiesis

The molecular mechanism(s) by which commensal microbes promote steady-state expansion

of bone marrow- and yolk sac-derived myeloid cells remains unknown. Microbial associated

molecular patterns (MAMPs) and microbial metabolites, such as short chain fatty acids

(SCFAs) have been shown to modulate various aspects of the host immune response (Chu

and Mazmanian, 2013; Clarke et al., 2010; Smith et al., 2013). Furthermore, MyD88 (an

adaptor for recognition of many MAMPs) was recently shown to promote GMP expansion

and differentiation (Fiedler et al., 2013). Accordingly, we sought to address whether

commensal-derived factors are involved in the maintenance of myeloid cells under naïve

conditions. Re-colonization of GF mice with a complex microbiota andoral treatment with

MAMPs, but not SCFAs, was sufficient to promote recovery of GMP-derived myeloid cells

(neutrophils and monocytes) within the bone marrow (Figure 4A, B). Importantly, only re-

colonization of GF mice with an SPF microbiota was sufficient to restore splenic

populations of F4/80

macrophagesand F4/80

splenocytes (i.e., neutrophils, monocytes

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and macrophages) (Figure 4C and Figures S4A-D). Therefore, while MAMP treatmentis

necessaryfor the maintenance of bone marrow-derived myeloid cells, colonization with a

live and complex microbiota is required to promote complete myelopoiesis (including yolk

sac-derived macrophages). Finally, only re-colonization of GF animals, and not oral MAMP

treatment, was sufficient to restore the defect in GF mice to systemic challenge with

monocytogenes

(Figure 4D and data not shown).Collectively, these studiesreveal that the

microbiota provides complex molecular signals that actively promote the hematopoietic

differentiation of myeloid cells, resulting in peripheral phagocyte populationsthat function as

sentinels for the early detection and control of systemic bacterial infection.

Discussion

Advances in understanding host-microbial symbiosis have revealed that, remarkably, the gut

microbiota controls the phenotype, migration and activity of multiple innate and adaptive

cells(Belkaid and Naik, 2013; Chu and Mazmanian, 2013). Disruption or alteration of

commensal communities impacts host susceptibility to various disorders, particularly at sites

of microbial colonization such as the intestines, respiratory mucosa and skin

epithelium(Kamada et al., 2013). In addition to modulating functional immune outcomes,

the microbiota is necessary for maintaining systemic populations of neutrophils in the

circulation and CD4

T cells in the spleen(Bugl et al., 2013; Mazmanian et al., 2005),

suggesting a possible contribution by gut microbiota to the development of the immune

system. Herein, we revealthat gut bacteria regulatehematopoiesiswithin primary immune

sites, providing a unifying explanation for previous observations of the widespread effectsby

the microbiota on the immune system. Specifically, our study uncovers that the microbiota

promotes steady-state myeloid cell development by driving the expansion of yolk sac-

derived macrophages, as well as enhancing the numbers and differentiation potential of

GMPs in the bone marrow. We propose a model whereby a primary defect in hematopoiesis

in GF or antibiotic-treated mice compromisesmultiple tissue-resident innate immune cell

populations prior to infection, leading to blunted early responses upon subsequent pathogen

encounter (see diagram in Figure S4E). While our studies focus on innate immunity due to

its role in rapid control of early

Listeria

infection, impaired microbiota-mediated

hematopoiesis may alsoextend to the adaptive immune system, providing an explanation for

observations that peripheral T, B and iNKT cell populations are altered in GF mice (Ivanov

et al., 2008; Macpherson and Uhr, 2004; Mazmanian et al., 2005; Olszak et al., 2012).

How commensal microbes (presumably in the gut) are able to control immune responses in

distant sites such as the bone marrow remains incompletely understood. It has recently been

shown that mice deficient in MyD88 signaling display reductions in systemic myeloid cell

populations and GMP numbers(Fiedler et al., 2013; Yanez et al., 2013), similar to our

findings in GF mice. Further, as microbial ligands have been detected in systemic sites,

including the bone marrow (Clarke et al., 2010), commensal-derived MAMPs that originate

in the gut may mediate steady-state myelopoiesis in primary immune sites. Accordingly, we

show that oral treatment with MAMPs is sufficient to rescue GMP-mediated expansion of

neutrophils and monocytes. However, MAMP treatment aloneis inadequate to expand

splenic F4/80

and F4/80

cells, indicating additional commensal-derived signals are

necessary to influence site-specific HSC and yolk sac-derived myeloid cells. Interestingly,

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re-colonization of adult GF mice with SPF microbiota is insufficient to restore splenic

F4/80

macrophages to the levels found in SPF mice. This may suggest that complete

rescue requires either colonization from birth or colonization with specific microbes that

were not transferred into GF mice. In addition to MAMPs translocating from the gut into the

circulation, other explanations for how the microbiota affects hematopoiesis may include a

role for myeloid cell growth factors. In support of this notion, preliminary data suggest that

GF mice are reduced in M-CSF transcript levels in the gut (data not shown), though further

work is need to uncoverthe complex molecular mechanism(s) by which commensal bacteria

signal from the gut to distant primary immune organs.

Finally, we speculate that these findings may be relevant to human infections. The spread of

antibiotic-resistance among pathogens, paired with a dwindling supply of effective

antibiotics,has necessitated alternative strategies to combatinfections (Khosravi and

Mazmanian, 2013). Evidence that depletion of the microbiota leads to transient immune

suppression suggests factors that disrupt commensal microbes, including clinical antibiotic

use may, paradoxically, be a risk factor for susceptibility to opportunistic pathogens. The

concepts proposed herein, if validated in humans, may herald future medical approaches that

combine antibiotics with immunomodulatorymicrobial molecules as revolutionary

combination treatments to address the reemerging crisis of infectious diseases.

Experimental Procedures

Animal Studies

Specific pathogen-free (SPF) C57BL/6 mice were purchased from Taconic Farms. Germ-

free (GF) C57BL/6 and C57BL/6 Rag

-/-

mice were bred and raised in sterile gnotobiotic

flexible film isolators at the California Institute of Technology. Mice at 8-12 weeks of age

were infected via retro-orbital injection with 3×10

colony forming units (CFU) of

Listeriamonocytogenes

10403S. Splenic and liver bacterial CFU were assessed 24-72 hpi by

microbiological plating. For microbiota depletion studies, SPF mice were treated with 1

mg/ml of ampicillin (Auromedics), neomycin sulfate (Fisher), streptomycin (Sigma) and 0.5

mg/ml of vancomycin (Sagent) in the drinking water for 4-5 weeks. Mice were taken off

antibiotics 4 days prior to infection. Antibiotic-treated and untreated SPF mice were infected

with 3×10

CFU of

L. monocytogenes

and splenic bacterial burden was assessed 72 hpi. GF

mice were recolonized by gavage with cecal contents of SPF mice. Alternatively, GF mice

were treated with MAMPs through the addition of heat killed

Escherichia coli

strain

Nissle(Lodinova-Zadnikova and Sonnenborn, 1997)or autoclaved cecal contents from SPF

mice in water (

∼

1×10

CFU/ml indrinking water). For treatment with short chain fatty

acids,sodium proprionate (Sigma), sodium butyrate (Sigma), and sodium acetate (Sigma)

was added to drinking water at previously described concentrations (25mM, 40mM and

67.5mM, respectively)(Smith et al., 2013). Mice were re-colonized or treated with microbial

ligands or metabolites for 4 weeks prior to cellular analysis and infectious studies. Animals

were cared for under established protocols and IACUC guidelines from the California

Institute of Technology.

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Cellular Analysis

Spleens were either mechanically disrupted viapassage through 100 μm mesh filters (BD

Biosciences) or digested in 0.5 mg/ml of Collagenase D (Roche) and 0.5 mg/ml of DNase I

(Worthington). Bone marrow was collected by flushing femurs with PBS containing 0.5%

BSA and 5mM EDTA. Single cell suspensions were removed of red blood cells (RBC lysis

buffer, Sigma). Mature myeloid cells were evaluated by staining with antibodies to GR1

(RB6-8C5), Ly6C (HK 1.4), CD11b (M1/70), CD115 (AFS98) and F4/80 (BM8). Mouse

hematopoietic stem and progenitor cells (HSPCs) were isolated from bone marrow by a

combination of MACS magnetic bead purification (Miltenyi) and fluorescence activated cell

sorting (FACS). Lineage marker-negative cells (Lin

) were first separated using a MACS

lineage cell depletion kit (containing antibodies against CD5 (53-7.3), CD45R (B220;

RA3-6B2), CD11b, Gr-1, 7-4 (15BS) and Ter-119 (Ter-119)) and an autoMACS Separator

(Miltentyi). Lin

cells were then further stained with c-Kit (CD117; 3C1), Sca-1 (D7),

CD16/CD32 (93), CD34 (RAM34). Populations of LKS

cells (Lin

c-Kit

Sca-1

; HSCs

and MPPs), Lin

c-Kit

Sca-1

(LKS

) CD34

cells (CMPs) and LKS

CD34

cells (GMPs) were analyzed by flow cytometry. LKS

CD34

cells were FACS

sorted using an Aria cell sorter (BD Biosciences). Steady-state cell proliferation was

measured by intraperitoneal (

i.p.

) injection of 500 μg EdU (Life Technologies) and EdU

incorporation among splenic myeloid cells was measured 24 hours later via Click-it EdU

assay kit (Life Technologies). Antibodies were purchased from eBioscience, BD Bioscience,

Miltenyi or Biolegend. Data were collected on a FACSCalibur or LSR Fortessa (BD

Bioscience) and analyzed with FlowJo software (TreeStar).

Cell Depletion and Adoptive Transfer

Resident phagocytes were depleted by intravenous(

i.v.

) treatment with 100 μl of clodronate-

loaded liposomes (CL; FormuMax) 48 hours prior to infection. CD11b

splenocytes were

isolated from naïve SPF and GF mice using CD11b microbeads (Miltenyi). 2×10

CD11b

cells (>90% purity) were transferred into GF recipients, 24 hours prior to infection with

monocytogenes

. CFU burden was assessed 24 hpi.

CFU Assays

To evaluate hematopoietic potential, 1×10

Lin

or 1×10

LKS

CD34

cells or

2×10

splenocytes were plated in triplicate in MethoCult GF M3434 (StemCell

Technologies) methylcellulose-based medium and incubated for 7 days in 37°C with 5%

CO2, after which the colonies were counted on the basis of their morphological

characteristics in accordance with the manufacturer's instructions. On the same day, cells

were harvested, counted and stained for c-Kit and CD11b expression for progenitor

quantification by flow cytometry. For re-plating assays, 5×10

cells from the first culture

were plated in triplicate in a secondary culture of fresh MethoCult GF M3434, and colonies

were counted after 7 days of incubation.

Supplementary Material

Refer to Web version on PubMed Central for supplementary material.

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Acknowledgments

We thank H. Chu, Y. K. Lee, G. Sharon, A. Nanz (Caltech), and N. Hassanzadeh-Kiabi (Cedars-Sinai) for technical

assistance, T. Thron for animal care, and S. McBride, D. Majumdar, S. Damle, P. Mehrabian (Caltech) and

members of the Mazmanian laboratory for critical reading of the manuscript. We thank U. Sonnenborn

(Ardeypharm GmbH) for the generous gift of

Escherichia coli

Nissle 1917. A.K. was supported by a pre-doctoral

training grant from the National Institute of Health (GM007616). This research was supported by a Burroughs

Wellcome Fund in the Pathogenesis of Infectious Disease award to S.K.M.

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Highlights

•

Germ-free mice are deficient in spleen and bone marrow resident myeloid cell

populations

•

Gut microbes impact both yolk sac- and stem cell-derived myeloid cell

development

•

Microbiota promotes early resistance to systemic

Listeria monocytogenes

infection

•

Re-colonizationrestores immune integrity against systemic Listeriosis in germ-

free mice

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Figure 1. GF Mice Are Deficient in Resident Myeloid Cell Populations in the Spleen and Bone

Marrow

(A-C) Splenic phagocyte profile among SPF and GF mice. Representative flow cytometry

plots (A), cell proportions (B), and total cell number (C) of CD11b

F4/80

and CD11b

F4/80

splenic cells in SPF and GF mice. (D-F) Bone marrow populations of neutrophils

(Gr1

CD115

neg

) and monocytes (Gr1

CD115

) among SPF and GF mice. Representative

flow cytometry plots (D), cell proportions (E) and total cell number (F) within the bone

marrow of SPF and GF mice. For all panels, data are representative of at least 3 independent

trials with n≥ 4 mice / group. Each symbol represents data from a single animal. Error bars

represent standard error of mean (SEM). *

<0.05, **

<0.01. PMN: polymorphonuclear

cells; Mono: monocytes. See also Figure S1.

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Figure 2. The Microbiota Directs Myelopoiesis

The percentage of F4/80

CD11b

(A) and F4/80

CD11b

(B) splenocytes with

incorporated EdU, following single dose administration. (C) The frequency of LKS

CD34

granulocyte and/or monocyte progenitors (GMPs) among lineage negative (Lin

)

progenitors from bone marrow of SPF and GF mice, as assessed by flow cytometry. (D)

Distribution of cell types following purified LKS

CD34

cell culture in

methylcellulose medium. Colonies were identified and counted to assess the proportion of

granulocyte-monocytes (GM-CFU; black), granulocytes (G-CFU; blue) and monocytes (M-

CFU; green). (E) Total numbers of c-Kit

CD11b

progenitors from methylcellulose cultures

of LKS

CD34

progenitors, as assessed by flow cytometry. (F) Cells harvested

from methylcellulose cultures of LKS

CD34

progenitors were re-plated at equal

numbers in fresh methylcellulose, and cultured to assess their colony forming capacity. (G

and H) Splenic cells isolated from SPF and GF mice were cultured in methylcellulose to

assess the colony forming capacity of progenitors from SPF and GF mice. Total CFUs (G),

and GM-CFUs, G-CFUs and M-CFUs (H) are shown. For each panel, data are

representative of at least 2-3 independent trials with n≥ 4/ group. Each symbol represents

data from a single animal. Error bars represent SEM. *

<0.05 for all panels. **

<0.05

(comparing total CFU between SPF and GF for (D) and (H)), ***

<0.05 (comparing G-CFU

between SPF and GF for (D) and (H)), ****

<0.05 (comparing M-CFU between SPF and

GF for (D) and (H)). CFU: colony forming units. See also Figure S2.

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Figure 3. The Microbiota Promotes Early Resistance to Systemic Infection by

L. monocytogenes

via Tissue-Resident Cells

(A-C) SPF and GF mice infected with

L. monocytogenes

and assessed for survival (A) and

splenic bacterial burden at 24 (B) and 72 (C) hours post- infection (hpi). SPF mice treated

with antibiotics (Abx) and untreated controls (Ctl) were infected with

L. monocytogenes

and

splenic bacterial burden was measured 72 hpi (D). SPF and GF mice depleted of tissue-

resident cells prior to infection with

L. monocytogenes

and assessed for splenic bacterial

burden 24 hpi (E) and survival (F). Splenic bacterial burden, 24 hpi, following transfer of

splenic CD11b

cells from SPF or GF donors (G). For all panels, data are representative of

at least 2-3 independent trials with n≥ 4/ group. Each symbol represents data from a single

animal. Error bars represent SEM. *

<0.05, **

<0.01, ***

<0.05 log-rank test used for

survival curves in (A). CL: clodronate-loaded liposomes. See also Figure S3.

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