, 597 (2012);335 Science
, et al.Philipp J. Rauch
Innate Response Activator B Cells Protect Against Microbial Sepsis
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Innate Response Activator B Cells
Protect Against Microbial Sepsis
Philipp J. Rauch,1* Aleksey Chudnovskiy,1* Clinton S. Robbins,1*† Georg F. Weber,1
Martin Etzrodt,1Ingo Hilgendorf,1,6Elizabeth Tiglao,1Jose-Luiz Figueiredo,1Yoshiko Iwamoto,1
Igor Theurl,1,3,7Rostic Gorbatov,1Michael T. Waring,4Adam T. Chicoine,4Majd Mouded,5
Mikael J. Pittet,1Matthias Nahrendorf,1Ralph Weissleder,1,2Filip K. Swirski1†
Recognition and clearance of a bacterial infection are fundamental properties of innate immunity.
Here, we describe an effector B cell population that protects against microbial sepsis. Innate
response activator (IRA) B cells are phenotypically and functionally distinct, develop and diverge
from B1a B cells, depend on pattern-recognition receptors, and produce granulocyte-macrophage
colony-stimulating factor. Specific deletion of IRA B cell activity impairs bacterial clearance, elicits
a cytokine storm, and precipitates septic shock. These observations enrich our understanding of
innate immunity, position IRA B cells as gatekeepers of bacterial infection, and identify new
treatment avenues for infectious diseases.
incidence of sepsis has risen, indicating the need
epsis is characterized by whole-body in-
flammation in response to overwhelming
infection (1). Over the past 30 years, the
for a better understanding of its complex patho-
physiology (2, 3). The growth factor granulocyte-
macrophage colony-stimulating factor (GM-CSF)
nate receptor. Yet, despite GM-CSF’s multiple
functions and known relationship with innate leu-
kocytes, its in vivo cellular source and role in
sepsis remain uncertain (4).
etry led to a surprising observation. Among the
organs, the bone marrow and spleen contained
and Harvard Medical School, Boston, MA 02114, USA.2De-
partment of Systems Biology, Harvard Medical School, Boston,
MA 02115, USA.3Program in Membrane Biology and Division
of Nephrology, Massachusetts General Hospital and Harvard
Medical School, Boston, MA 02114, USA.4Ragon Institute
Imaging Core, Massachusetts General Hospital and Harvard
Medical School, Boston, MA 02114, USA.5Division of Pul-
monary, Allergy, and Critical Care Medicine, University of
Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA.
6Department of Cardiology, University Hospital Freiburg,
79106 Freiburg, Germany.7Department of General Internal
Medicine, Clinical Immunology and Infectious Diseases, Uni-
versity Hospital of Innsbruck, A-6020 Innsbruck, Austria.
*These authors contributed equally to this work.
†To whom correspondence should be addressed. E-mail:
firstname.lastname@example.org (F.K.S.); robbins.clinton@mgh.
Fig. 1. IRA B cells are GM-CSF–producing B cells that increase in number
during inflammation. (A) Quantification of GM-CSF–producing cells retrieved
from tissues in the steady state and in response to four daily intraperitoneal
injections of LPS (means T SEM, n = 3 to 5 mice). *P < 0.05. (B) Iden-
tification of GM-CSF–producing cells in the spleen. Representative plots
show percentage of B cells and their production of GM-CSF retrieved from
spleens during inflammation. Data represent at least 10 independent exper-
iments. (C) Western blot for GM-CSF conducted on sorted cells. One of three
independent experiments is shown. GAPDH, glyceraldehyde-3-phosphate
dehydrogenase. (D) Colocalization of representative GM-CSF–producing
cells with IgM. (E) Red pulp sections with markers against CD11b (green)
and GM-CSF (red) (left panel) and B220 (green) and GM-CSF (red) (right
panel). Colocalization of green and red cells is shown in yellow. The dashed
white curve indicates the border between white and red pulp. (F) Quan-
tification of GM-CSF+B cells and other cells on histological sections of
the spleen in the red pulp and white pulp in the steady state and after LPS
(means T SEM, n = 3 to 4). *P < 0.05. (G) Splenic GM-CSF expression
detected by RT-PCR and conducted on sorted cells and on unprocessed
spleen tissue taken from WT and B cell knockout (mMT) mice (means T SEM,
n = 3 to 4). *P < 0.05.
VOL 335 3 FEBRUARY 2012
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(1.0 T 0.1 × 106and 2.9 T 0.8 × 105cells, respec-
tively) (Fig. 1A) (5). In response to lipopolysac-
charide (LPS), a component of Gram-negative
erentially in the spleen (3.2 T 0.2 × 106cells) and
B cells (IgM, immunoglobulin M) (Fig. 1B and
CSF is believed to be produced in vivo by non-
hematopoietic cells, macrophages, and, in some
cases, Tcells (4, 6). Nevertheless, B cells consti-
by Western blot analysis (Fig. 1C). We named
these B cells innate response activator (IRA) B
ing innate leukocytes. Numerous IRA B cells ac-
cumulated in the spleen in a mouse model of
sepsis (fig. S2, A and B) (7) and in response to
Escherichia coli infection (fig. S2C), indicating
that IRA B cell expansion is a general feature of
the body’s response to bacteria. In humans, we
detected CD19+CD20+IRA B cells expressing
(8). We therefore elected to characterize murine
IRA B cells in more detail.
Immunofluorescence of spleen sections from
LPS recipients colocalized the GM-CSF signal
with round mononuclear cells expressing IgM,
B220, PAX5, and CD19 (Fig. 1D and fig. S1D)
in the red pulp (Fig. 1, E and F). Reverse tran-
scription polymerase chain reaction (RT-PCR)
experiments conducted on sorted cells and un-
processed tissue from wild-type (WT) or B cell–
GM-CSF (Fig.1G). Serum GM-CSF levels were
negligible (that is, below the 7.8 pg/ml detection
limit of the assay), a result that is consistent with
the observation that GM-CSF is rapidly removed
ively, these data indicate that inflammation ex-
pands the IRA B cell population in vivo.
B cells are linked developmentally, reside in
different regions, and mediate distinct functions
(10–14). We profiled IRA B cells according to
several well-established methods (13, 15, 16).
Our experiments revealed that (CD19+B220+
MHCII+GM-CSF+) IRA B cells are phenotypi-
CD93+(Fig. 2, A and B, and fig. S3A), IgDlow
CD21low(fig. S3B), CD138+VLA4highLFA1high
CD284+(Fig. 2C and fig. S3, C and D), and
CD5int(fig. S3, E and F). IRA B cells contain
large stores of intracellular IgM (fig. S4A) and
spontaneously secrete IgM, but not IgA or IgG1
Fig. 2. IRA B cells are a distinct subset with a unique phenotypic signa-
ture. (A) Flow cytometric analysis of the phenotype of IRA B cells. Plots
show B cell phenotypes retrieved from spleens during steady state and
inflammation. A representative from n > 10 mice is shown. (B) Plots show
the phenotype of GM-CSF–producing cells in the spleens during in-
flammation. IRA B cells are IgMhigh, CD23lowCD43+CD93+. (C) Plots
show the phenotype of IRA B cells with respect to VLA4 and CD138
expression as determined by flow cytometry. A representative from n>5
is shown. (D) Hierarchical clustering dendrogram based on whole-genome
microarray data of sorted samples of B cell subsets retrieved from LPS-
treated animals and steady-state B1a. (E) PCA of the different cell subsets
shown in (D).
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(fig. S4,B and C).Inaddition toGM-CSF,IRA
B cells produce interleukin-3 (IL-3) but not
pro–IL-1b, IL-6, and tumor necrosis factor–a
(TNFa) (fig. S4D). We failed to detect IL-10
expression by IRA B cells in any of the con-
ditions. Thus, IRA B cells have a unique B cell
phenotype and are functionally distinct from
other B cells, including the recently described
IL-10–producing B10 B cells (17).
Sorting IRAB cells according to theirsurface
phenotype (fig. S5A) allowed us to profile their
transcriptome. Unsupervised hierarchical clus-
tering (Fig. 2D) and principal component analy-
sis (PCA) (Fig. 2E) grouped IRA B cells in a
separate population from T1, FO, MZ, B1a, and
PC. IRA B cells also gave rise to a unique tran-
scriptome signature (fig. S5, B to D, and table
S1) and expressed genes relevant to B cell bi-
ology (fig. S5D).
lineage, we performed several parabiosis and
fate-mapping studies. First, we reasoned that if
they should have high chimerism in a parabiosis
setting. Joining CD45.1+with CD45.2+mice
revealed high chimerism among IRA B cells
(Fig. 3A), T1, and FO B cells (fig. S6A) but mark-
edly lower chimerism for the spleen-resident MZ
B cells derive from a circulating cell.
Second, to identify the IRA B cell precursor,
we adoptively transferred B cell subsets to mice
the subsets (splenic T1, FO, MZ, and B1 and
peritonealB1a,B1b,andB2), only peritonealB1
B cells (Fig. 3B) gave rise to IRA B cells. Of
Spleens of CD45.1+ mice that received CD45.2+ B cells
peritoneal B1a cells
LPS & VLA-4/LFA-1
CD45.2-Alexa700CD19+ cells (spleen)
Chimerism in CD45.1+ spleens
during parabiosis with CD45.2+ partner
splenic B cells
Fig. 3. IRABcellsdevelopfromB1aBcellsviaTLR4/MyD88andresideintissue
through LFA-1/VLA-4. (A) Flow cytometric analysis of the percent chimerism is
shown in spleens of CD45.1+mice that had been in parabiosis with CD45.2+
mice for 3 weeks before LPS injection. Mice were sacrificed 2 days after LPS
Adoptive transfer of peritoneal B1a B cells yields IRA B cells. Cells from steady-
stateCD45.2+mice were transferred toCD45.1+mice that then received LPSfor
of the development of IRA B cells in Tlr4–/–, Myd88–/–, Ticam1–/–(the gene that
encodes TRIF), mMT, Tnfrsf13c–/–(the gene that encodes BAFFR), and Cd19–/–
cells in steady state and inflammation in WT (C57BL/6) mice and in the mice
analysis of the effect of blocking VLA-4/LFA-1 on IRA B cell retention in the
spleen. A representative from n = 3 mice is shown.
VOL 3353 FEBRUARY 2012
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B1a-derived IRA B cells readily proliferated
(fig. S6E) and developed in the spleen after re-
locating from the peritoneum (fig. S7). These
findings confirm that B1a B cells travel to the
spleen in response to peritoneal TLR stimuli
(18, 19) and indicate that, upon splenic accu-
mulation, B1a B cells can differentiate to IRA
The ontogenic relationship between B1a
and IRA B cells raised the question of whether
IRA B cells constitute a distinct subset. To elu-
cidate this, we first placed peritoneal B1a B cells
in culture. In response to LPS, B1a B cells sep-
arated into three discrete populations: CD138–
cells resembling “unchanged” B1a B cells and
two populations of CD138+cells, one of which
was IRA B cells (fig. S8A). In vitro, IRA B cells
spontaneously secreted GM-CSF (fig. S8B). We
then sorted peritoneal B1a B cells, IRA B cells,
and splenic CD43+CD138+cells and followed
their fate in vivo. B1a B cells gave rise to mul-
tiple cell types (fig. S9A), including IRA B and
CD43+CD138+cells, whereas (CD43highCD138+)
IRA B and CD43+CD138+cells remained phe-
notypically segregated (fig. S9, B and C). The
data suggest that B1a B cells give rise to distinct
cells. IRA B cells are a subset of this group.
Surface phenotype and fate-mapping studies,
though important, reveal little about function.
How IRA B cells arise was our next question.
(21) mice did not develop IRA B cells (Fig. 3,
the B cell–activating factor receptor (BAFFR)
failed to generate IRA B cells; BAFFR is be-
level of microbial recognition, mice lacking the
LPS receptor TLR4 or its adaptor MyD88, but
Fig. 4. IRA B cells protect against polymicrobial sepsis. (A) Generation of
mixed chimeras (GM/mMT). gy, gray; wk, weeks. (B) Kaplan-Meier curve show-
ing survival of GM/mMT and control animals after CLP. n = 10 to 20 mice per
group. (C) Enumeration of total leukocytes and neutrophils in the peritoneum
of GM/mMT (dark red bars) and control (black bars) mice 20 hours after CLP.
W, steady state. *P < 0.05. (D) Serum levels and (E) peritoneal levels of in-
flammatory cytokines in GM/mMT (dark red) and control (black) mice 20 hours
after CLP. (F) Ex vivo phagocytosis assay showing capacity of neutrophils to
phagocytose E. coli from GM/mMT (dark red) and control (black) mice 20 hours
after CLP. (G) Serum levels of IgM and IgG 20 hours after CLP in the same
groups as above. (H) Representative hematoxylin and eosin stain of liver and
lung sections 20 hours after CLP in the same groups as above. (I) Blood from
GM/mMT and control mice 20 hours after CLP was plated for 1 day. Rep-
resentative plate shows bacterial colonies. (J) Enumeration of bacteremia
intheperitoneumandbloodofGM/mMT(darkred) andcontrol(black) mice
20 hours after CLP. *P < 0.05 [means T SEM, n = 10 to 20 mice per group for
(C) to (G) and (J). Four independent experiments were performed and data
3 FEBRUARY 2012VOL 335
on February 2, 2012
not TRIF, did not generate IRA B cells (Fig. 3, C Download full-text
and D), indicating a specific MyD88-dependent
pathway. The process could depend on direct
B1a binding to LPS via TLR4, or on indirect,
extrinsic factors such as TLR4-expressing macro-
phages. To discriminate between these two pos-
sibilities, we adoptively transferred B1a B cells
from WT mice into Tlr4–/–mice (Fig. 3E). B1a
WT B cells, but not endogenous Tlr4–/–B cells,
differentiated to IRA B cells, indicating that di-
rect TLR4 signaling on B1a B cells is sufficient
to generate IRA B cells.
To test whether IRA B cells are restricted to
ands Pam3CSK4 (ligand for TLR1/2), Poly(I:C)
(TLR7/8), and CpG ODN1668 (TLR9). The lig-
ands Pam3CSK4, FSL-1, and R848 yielded IRA
B cells (fig. S10A), a finding that we confirmed
in vitro (fig. S10B). We also wondered whether
(CD131) (fig. S11A) and, when placed in culture
to IRA B cells (fig. S11, B and C) but remained
alive and gave rise to CD43+CD138+cells. Thus,
ways and use GM-CSF as an autocrine factor.
The spleen’s open circulation (24) allows
blood leukocytes to enter and exit easily. To re-
side in the spleen, leukocytes resort to adhesive
ligands; MZ B cells,for example, rely on VLA-4
and LFA-1 (25). We wondered whether splenic
IRA B cells,which express VLA-4 and LFA-1 at
high levels, might behave similarly. Injection of
neutralizing antibodies toVLA-4 and LFA-1 di-
minished IRA B cell numbers and revealed that
the two integrins are responsible for retention
Are IRA B cells functionally important? To
answer this, we focused on the cecal ligation and
puncture (CLP) sepsis model (26). We generated
mixed chimeras by reconstituting lethally irra-
diated mice with mMT- and GM-CSF–deficient
GM/mMT chimeras), the mMT marrow contrib-
uted all leukocytes except B cells, whereas the
Csf2–/–marrow contributed only Csf2–/–cells.
Consequently, the only population completely
lacking the capacity to produce GM-CSF in the
reconstituted mice were B cells. We tested the
quality of the chimeras and their controlsbyPCR
(fig. S11, A and B) and by flow cytometry (fig.
S11, C and D).
In response to severe CLP, 40% of control
chimera died within 2 days (Fig. 4, A and B).
To characterize this phenotype further, we pro-
filed GM/mMTchimeras and controls for several
sepsis-relevant indices 20 hours after CLP, be-
fore any mortalities. Compared with IRA B cell–
containing controls (fig. S11E), the peritoneal
cavities of GM/mMT chimeras had more leuko-
cytes, mostly neutrophils (Fig. 4C), and experi-
enced a severe IL-1b, IL-6, and TNFa cytokine
storm in the serum (Fig. 4D) and peritoneum
(Fig. 4E). This inflammatory signature typically
associates with a defect in bacterial clearance.
Indeed, neutrophils from the GM/mMTchimeras
phagocytosed bacteria poorly (Fig. 4F). More-
over, the GM/mMT chimeras had a modest re-
developed severeliverand lung pathologies (Fig.
that GM/mMTchimeras were more infected than
controls (Fig. 4, I and J). Although it is possible
most likely explanation is that IRA B cells pro-
tect against septic shock by controlling the orga-
nism’s ability to clear bacteria.
GM-CSF is a pleiotropic cytokine that influ-
ences the production, maturation, function, and
survival of its target cells. GM-CSF’s role in sep-
can be beneficial (28). The in vivo identification
of GM-CSF–producing B cells illustrates a pre-
viously unrecognized locational specificity that
dictates the cytokine’s function. IRA B cells dif-
fer from other subsets because their pathogen
recognition pathways and tissue distribution li-
cense GM-CSF expression. The function is im-
portant in sepsis and gives rise to questions as to
how IRA B cells participate in other infectious
and inflammatory diseases.
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Acknowledgments: This work was supported in part by
NIH grants 1R01HL095612 (to F.K.S.), as well as U01
HL080731, P50 CA86355, R24 CA69246, and P01-A154904
(to R.W.). P.J.R. was supported by the Boehringer Ingelheim
Fonds; C.S.R. was supported by an American Heart Association
postdoctoral fellowship and the MGH Executive Committee on
Research (ECOR) Postdoctoral Award; G.F.W. and I.H. were
supported by the German Research Foundation; and I.T. was
supported by the Max Kade Foundation. We thank K. Rajewsky,
D. Scadden, A. Luster, and K. Otipoby (Harvard Medical School)
for helpful discussions and critical reading of the manuscript
and M. Greene for secretarial assistance. The data reported
in this paper are tabulated in the main paper and in the
supporting online material. MIAME (minimum information
about a microarray experiment)–compliant expression data
have been deposited under the accession no. GSE32372.
Supporting Online Material
Materials and Methods
Figs. S1 to S12
11 October 2011; accepted 22 December 2011
Published online 12 January 2012;
Abnormal Brain Structure Implicated
in Stimulant Drug Addiction
Karen D. Ersche,1* P. Simon Jones,1Guy B. Williams,1,2Abigail J Turton,1
Trevor W. Robbins,1Edward T. Bullmore1,3,4
Addiction to drugs is a major contemporary public health issue, characterized by maladaptive
behavior to obtain and consume an increasing amount of drugs at the expense of the individual’s
health and social and personal life. We discovered abnormalities in fronto-striatal brain systems
implicated in self-control in both stimulant-dependent individuals and their biological siblings
who have no history of chronic drug abuse; these findings support the idea of an underlying
neurocognitive endophenotype for stimulant drug addiction.
rug dependence is increasingly recog-
nized as a “relapsing brain disorder” (1)
and, in support of this view, marked struc-
tural changes in striatal and prefrontal brain re-
gions have been reported in people dependent on
stimulant drugs (2). These reports, however, raise
VOL 3353 FEBRUARY 2012
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