Cell Host & Microbe, Volume
15
Supplemental Information
Gut Microbiota Promotes Hematopoiesis
to Control Bacterial Infection
Arya Khosravi, Alberto Yáñez, Jeremy G. Price, Andrew Chow, Miriam Merad,
Helen S. Goodridge, and Sarkis K. Mazmanian
SUPPLEMENTAL
FIGURES
Figure S1. GF and Antibiotic
-
Treated Mice Have Reduced Populations of Myeloid Cells in
Systemic Sites, Related to Figure 1
(A) Frequency of splenic neutrophils (CD11b
+
GR1
hi
Ly6c
lo
), monocytes (CD11b
+
Ly6c
hi
GR1
hi
) and macrophages (CD11b
+
GR1
-
F4/80
lo
) among SPF
and GF mice. (B) Frequency of splenic CD11b
+
F4/80
hi
and CD11b
+
F4/80
lo
phagocytes among
untreated mice (Ctl) and
SPF mice treated with oral antibiotics (Abx). (C) Frequency of liver
CD11b
+
F4/80
hi
macrophages recovered from SPF or GF mice. Error bars represent
standard
error of mean (
SEM
)
. Data are representative of 2
-
3 independent trials with n
≥
4
/ group
.
*
p
<0.05, **
p
<0.01. PMN: polymorpho
nuclear cells; Mono: monocytes; M
Ф
: macrophages.
Ctl
Abx
Ctl
Abx
0
2
4
6
8
10
F4/80
hi
F4/80
lo
*
**
Total Cells (%)
SPF
GF
0.0
0.5
1.0
1.5
2.0
2.5
**
Total Cells (%)
SPF
GF
SPF
GF
SPF
GF
0
1
2
3
4
5
PMN
Mono
**
**
M
Φ
**
Total Cells (%)
A
B
C
Figure S2. GF Mice Have Normal Proportions and Differentiation Potential of HSCs and
Early Myeloid Progenitors in the Bone Marrow, Related to Figure 2
(A) Proportion of LKS
+
cells (Lin
-
c
-
Kit
+
Sca
-
1
+
;
HSCs and MPPs), (B) LKS
-
cells (Lin
-
c
-
Kit
+
Sca
-
1
-
; lineage
-
restricted
progenitors) and (C) CMPs (LKS
-
CD34
+
Fc
γ
R
lo
) among total progenitors (Lin
-
cells) of SPF and
GF mouse bone marrow. (D
-
F) Unfractionated bone marrow progenitor cells (Lin
-
cells) from
SP
F and GF mice cultured in methylcellulose to assess the colony forming potential of
progenitors. (D) E
-
CFU; erythrocyte colony forming units, (E) Meg
-
CFU; megakaryocyte CFU,
(F) GEMM
-
CFU; Granulocyte/erythrocyte/monocyte/megakaryocyte CFU. Error bars repre
sent
SEM. Data are representative of 3 independent trials with n
≥
4
/ group
.
Error bars represent SEM.
ns: non
-
significant.
SPF
GF
0.00
0.05
0.10
0.15
ns
CMP
(% Lin
-
cells)
SPF
GF
0
2
4
6
8
ns
LKS
+
(% Lin
-
cells)
SPF
GF
0
20
40
60
80
ns
LKS
-
(% Lin
-
cells)
SPF
GF
0
2
4
6
ns
E-CFU
SPF
GF
0
1
2
3
4
ns
Meg-CFU
SPF
GF
0
20
40
60
80
100
ns
GEMM-CFU
A
B
C
D
E
F
Figure
S3. Resident Phagocytes Mediate
Commensal
-
Enhanced Protection Against
Infectious Disease, Related to Figure 3
(A) SPF and GF mice infected with
L. monocytogenes
, liver bacterial burden assessed 72 hpi. (B)
SPF and GF mice infected with
S. aureus.
Kidney bacterial burden assessed 5 days post
-
infection. (C) Peritoneal macrophages isolated from SPF or GF mice, untreated or stimulated
with interferon
-
γ
(IFN
γ
), infected with
L. monocytogenes
. Recovery of intracellular bacteria
measured over time. Data
is non
-
significant for all time points measured, except where indicated
(untreated SPF vs. GF, 4 hpi). (D) SPF and GF Rag
-
/
-
mice infected with
L. monocytogenes
,
splenic bacterial burden assessed 72 hpi. (E) SPF and GF mice were immunized with
L.
monocytog
enes
Δ
actA
. 45 days after immunization, SPF and GF mice, as well as naïve, non
-
immunized SPF controls, were infected with wild
-
type (WT)
L. monocytogenes
. Splenic bacteria
burden of the WT strain was measured at 72 hpi. Note: two of the four naïve, non
-
imm
unized
SPF mice died following infection, prior to the 72 hour time point (data not shown). (F) BrdU
incorporation among bone marrow neutrophils (CD11b
+
GR1
hi
) and monocytes (CD11b
+
CD115
+
), 72 hpi. (G) Percentage of splenic neutrophils (Gr1
hi
Ly6C
lo
) and
monocytes (Gr1
hi
SPF
GF
SPF
GF
0
20
40
60
PMN
Mono
ns
ns
% BrdU+
SPF
GF
SPF
GF
0
5
10
15
20
PMN
Mono
**
*
Cells (%)
Rag
-/-
SPF
GF
4
5
6
7
8
9
**
CFU (Log)
SPF
GF
0
20
40
60
80
*
Total CFU (x10
6
)
SPF
GF
SPF
GF
4
6
8
10
α
Ly6G
**
**
Ve hicle
CFU (Log)
SPF
GF
SPF
GF
4
5
6
7
8
WT
CCR2
-/-
**
**
CFU (Log)
B
C
A
D
F
G
E
H
SPF
GF
2
4
6
8
**
CFU (Log)
SPF
GF
Naive
0
2
4
6
8
ns
CFU (Log)
SPF
GF
0
10
20
30
40
*
Annexin V+ (%)
I
0
2
4
6
8
0
100
200
300
SPF
GF
SPF + IFN
γ
GF + IFN
γ
*
Time (hrs)
Total CFU (x10
3
)
J
Ly6C
hi
) among SPF and GF mice, 72 hpi. (H) Annexin V
+
bone marrow monocytes, 72 hpi. (I)
SPF and GF mice infected with
L. monocytogenes
, following neutrophil depletion. Splenic
bacterial burden assessed at 72 hpi. (J) Splenic bacterial bu
rden of SPF and GF mice,
reconstituted with bone marrow from WT or CCR2
-
/
-
mice, 72 hpi. SPF mice reconstituted with
CCR2
-
/
-
bone marrow
display a two
-
fold reduction in splenic CFUs compared to GF CCR2
-
/
-
mice. For all panels, data are representative of 2
-
3 independent trials with n
≥
4
/ group
. Each
symbol represents data from a single animal. Error bars represent SEM. *
p
<0.05, **
p
<0.01.
PMN: polymorphonuclear cells; Mono: monocytes.
Figure S4. Re
-
colonization of GF Mice Rescues Tissue
-
Resident Phagocytes and Protects
Against Systemic Infection, Related to Figure 4
Percentage of F4/80
lo
splenocytes (A) as well
as splenic neutrophils (B), monocytes (C), and F4/80
lo
macrophages (D) among
SPF, GF, re
-
colonized GF and GF mice treated with MAMPs or SCFAs. (E) A proposed model for how the
microbiota mediates host resistance to systemic infection. Commensal microbes stimulate bone
marrow and splenic myelopoiesis during naïve conditions (in the
absence of infection),
expanding systemic pools of mature myeloid cells in SPF mice that are essential for restricting
pathogen dissemination upon acute infection. GF mice have reduced proportions and
differentiation pote
ntial by GMPs during the steady
-
st
ate, as well as diminished expansion of
yolk sac
-
derived macrophages, impairing the immune response to infection with
L.
monocytogenes
. This model suggests that conditions in which the microbiota is disrupted may
result in deficient expansion of myeloid ce
lls, compromising host resistance to infectious disease.
For all panels, data are representative of at least 2 independent trials with n
≥
4
/ group
. Error bars
represent
SEM
. *
p
<0.05. Recol: re
-
colonized; MAMPs: molecular associated
molecular patterns;
SCFAs: short chain fatty acids; PMN:
polymorphonuclear cells
; Mono: monocyte; M
Ф
:
macrophage.
+
SPF
GF
GF
GF
GF
2.0
2.5
3.0
3.5
4.0
Recol
MAMPs
-
-
+
-
*
SCFAs
-
-
-
-
-
-
-
-
+
-
*
ns
F4/80
lo
%
+
SPF
GF
GF
GF
GF
0.0
0.1
0.2
0.3
0.4
Recol
MAMPs
-
-
+
-
*
SCFAs
-
-
-
-
-
-
-
-
+
-
*
ns
PMN (%)
+
SPF
GF
GF
GF
GF
0.0
0.5
1.0
1.5
Recol
MAMPs
-
-
+
-
*
SCFAs
-
-
-
-
-
-
-
-
+
-
*
ns
Mono ( %)
+
SPF
GF
GF
GF
GF
1.0
1.5
2.0
2.5
Recol
MAMPs
-
-
+
-
*
SCFAs
-
-
-
-
-
-
-
-
+
-
*
ns
M
Φ
(%)
A
B
C
D
E