Interleukin-10 mediated autoregulation of murine B-1 B-cells and its role in Borrelia hermsii infection.

Abstract

B cells are typically characterized as positive regulators of the immune response, primarily by producing antibodies. However, recent studies indicate that various subsets of B cells can perform regulatory functions mainly through IL-10 secretion. Here we discovered that peritoneal B-1 (B-1P) cells produce high levels of IL-10 upon stimulation with several Toll-like receptor (TLR) ligands. High levels of IL-10 suppressed B-1P cell proliferation and differentiation response to all TLR ligands studied in an autocrine manner in vitro and in vivo. IL-10 that accumulated in cultures inhibited B-1P cells at second and subsequent cell divisions mainly at the G1/S interphase. IL-10 inhibits TLR induced B-1P cell activation by blocking the classical NF-kappaB pathway. Co-stimulation with CD40 or BAFF abrogated the IL-10 inhibitory effect on B-1P cells during TLR stimulation. Finally, B-1P cells adoptively transferred from the peritoneal cavity of IL-10(-/-) mice showed better clearance of Borrelia hermsii than wild-type B-1P cells. This study described a novel autoregulatory property of B-1P cells mediated by B-1P cell derived IL-10, which may affect the function of B-1P cells in infection and autoimmunity.

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Interleukin-10 Mediated Autoregulation of Murine B-1
B-Cells and Its Role in Borrelia hermsii Infection
Vishal Sindhava1,2, Michael E. Woodman1, Brian Stevenson1, Subbarao Bondada1,2*
1Department of Microbiology, Immunology and Molecular Genetics, University of Kentucky College of Medicine, Lexington, Kentucky, United States of America, 2Markey
Cancer Center, University of Kentucky College of Medicine, Lexington, Kentucky, United States of America
Abstract
B cells are typically characterized as positive regulators of the immune response, primarily by producing antibodies.
However, recent studies indicate that various subsets of B cells can perform regulatory functions mainly through IL-10
secretion. Here we discovered that peritoneal B-1 (B-1P) cells produce high levels of IL-10 upon stimulation with several Toll-
like receptor (TLR) ligands. High levels of IL-10 suppressed B-1P cell proliferation and differentiation response to all TLR
ligands studied in an autocrine manner in vitro and in vivo. IL-10 that accumulated in cultures inhibited B-1P cells at second
and subsequent cell divisions mainly at the G1/S interphase. IL-10 inhibits TLR induced B-1P cell activation by blocking the
classical NF-kB pathway. Co-stimulation with CD40 or BAFF abrogated the IL-10 inhibitory effect on B-1P cells during TLR
stimulation. Finally, B-1P cells adoptively transferred from the peritoneal cavity of IL-102/2mice showed better clearance of
Borrelia hermsii than wild-type B-1P cells. This study described a novel autoregulatory property of B-1P cells mediated by B-
1P cell derived IL-10, which may affect the function of B-1P cells in infection and autoimmunity.
Citation: Sindhava V, Woodman ME, Stevenson B, Bondada S (2010) Interleukin-10 Mediated Autoregulation of Murine B-1 B-Cells and Its Role in Borrelia hermsii
Infection. PLoS ONE 5(7): e11445. doi:10.1371/journal.pone.0011445
Editor: Olaf Rotzschke, Agency for Science, Technology and Research (A*STAR), Singapore
Received March 22, 2010; Accepted June 7, 2010; Published July 6, 2010
Copyright: ? 2010 Sindhava et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: These studies were funded by National Institutes of Health (NIH) grants CA09357, AI076956 and funds from the vice president for research at the
University of Kentucky to S. Bondada and exploratory funds from the University of Kentucky College of Medicine to Brian Stevenson. The funders had no role in
study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: bondada@uky.edu
Introduction
Regulation of the immune responseis as important as its activation
to prevent harmful effects caused by effector cells. Both cell intrinsic
(central tolerance) and cell extrinsic (regulatory cells) mechanisms
prevent the development of autoimmunity as well as negatively
control exaggerated immune responses [1,2]. Janewayand colleagues
were the first to demonstrate a regulatory role for B cells by
demonstrating that experimental autoimmune encephalomyelitis
(EAE) is enhanced in a B cell deficient environment [3]. B cells
that negatively regulate different immune responses through IL-10
production were termed ‘‘regulatoryB cells’’ by Mizoguchi and Bhan
[4]. Recent studies have shown that IL-10 producing B-cell subsets
with varying phenotypes can regulate different immune responses in
numerous mouse models, such as inflammatory bowel disease (IBD),
EAE, type 1 diabetes, collagen-induced arthritis, contact hypersen-
sitivity and during parasitic infection [5]. Despite the diversity of B
cellsubsetsinvolvedinthediseasemodels,theregulatorymechanisms
are uniformly dependent on IL-10 production. One of the high IL-10
producing subsets is the CD1dhiCD5+B cell subset, termed ‘‘B10
cells’’ by Yanaba and Tedder [6]. Matsushita et al. showed that
depletion of B cells with anti-CD20 antibodies before or during early
stages of EAE induction enhanced the disease [7]. B cell depletion
during the active disease period decreased the intensity of disease,
presumably due to the antigen presenting cell function of B cells. In a
clinical trial of B cell depletion therapy for ulcerative colitis, B cell
depletion exacerbated the disease [8].
Peritoneal B-1 (B-1P) cells were one of the first B cell subsets to
be identified to have the ability to produce IL-10. The B-1 cells
were described almost two decades ago and have recently been
shown to form a distinct B cell lineage [9]. The B-1 cell subset
expresses the pan T cell marker CD5 and is present in the spleen
as well as the peritoneal cavity. It is further subdivided into B-1a
and B-1b subsets based on differential expression of CD5 versus
Mac-1 [10]. The B-1a subset is required for production of natural
antibodies whereas the B-1b subset is involved in adaptive immune
responses to certain bacterial infections [11,12,13]. B-1P cells are
the source of natural IgM present in serum, mucosal IgA [10] and
play an important role in immunity against bloodborne pathogens
[12,14,15]. B-1 cells express antibody specificities against con-
served bacterial epitopes such as phosphorylcholine as well as self
antigens such as ssDNA, Thy1 and red blood cells. In humans,
rheumatoid factor producing B cells are present predominantly in
the B-1 subset [16]. Also, B-1 cells are elevated in several mouse
models of lupus [10]. B-1 cells proliferate poorly in response to
BCR crosslinking, presumably to protect against accidental
activation by self antigens [17,18,19]. This is in part due to
negative regulation by CD5 and in part due to defects in
generation of synergistic signals via B cell receptor (BCR) and
CD19 [17,20]. Despite the ability of B-1P cells to produce more
IL-10 than B-2 cells [21], a regulatory role for them has been
shown only in the IBD model [22].
Toll-like receptors (TLRs) are pattern recognition receptors that
recognize pathogen associated molecular patterns, which trigger
innate immunity leading to initiation of adaptive immunity.
Several B cell subsets express TLRs and can be activated via TLR
ligands which result in robust proliferation and antibody secretion,
even in the absence of dendritic cell activation or aid from T cells
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[23,24]. In addition to CD4+T cell help, generation of T-
dependent antigen specific antibody responses requires activation
of TLRs in B cells [25]. Although this is a controversial finding, it
appears to be dependent on the chemical modification of the
antigen [26,27]. TLR signals are also essential for T-independent
pathogen-specific IgM response [28]. B-1P cells require intact
TLR signaling for the clearance of Borrelia hermsii, which causes
relapsing fever in both humans and mice [28]. Furthermore, TLR
mediated signals synergize with self-antigen mediated BCR signals
to stimulate activation of self-reactive B cells [29].
In this study, we demonstrated for the first time that IL-10 plays
an auto-regulatory role in B-1P cells. B-1P cells produced much
higher levels of IL-10 than all the splenic B cell subsets both
constitutively and after stimulation with a variety of TLR ligands.
We also made the surprising observation that B-1P cells responded
to TLR ligands significantly less than splenic B-2 cells (B-2S) as
measured by proliferation and antibody production. High and
rapid IL-10 production by B-1P cells upon TLR stimulation
inhibited their own proliferation by blocking the classical NF-kB
pathway. Co-stimulation with CD40 and BAFF (B cell activating
factor belonging to the TNF family), but not IL-5, overcame the
IL-10 mediated inhibition of B-1 cells. We showed that high IL-10
production by B-1P cells hampered the clearance of B. hermsii in B-
1P transferred mMT mice.
Results
B-1P cells are hyporesponsive to TLR ligands
During the course of our studies to determine if TLR4 and B
cell receptor signals synergize in B-1P cells, we made the
surprising observation that B-1P cells are hyporesponsive to LPS,
a TLR4 ligand, mediated proliferation response when compared
to B-2S cells. Both B-1P and B-2S cells responded similarly to
CD40 mediated signaling (Fig. 1A). Several previous studies did
not emphasize such differences in the LPS response of B-1 and B-
2 cells, even though data in some reports supports our
observation [30,31,32]. The hyporesponsiveness of B-1P cells
was independent of the purity (protein or DNA free) of the LPS
used (Fig. 1A). On average, we found that the B-1 cell response to
LPS is 1766% of the B-2 cell response (n=7, p,0.004, with an
average of 5 mice in each experiment). Interestingly, B-1P cells
remained highly viable even up to 6 days after stimulation, while
viability of B-2S cells decreased significantly after two days of
stimulation, ruling out reduced viability as a cause of B-1P cell
hyporesponsiveness to LPS. In spite of this decrease in viability,
the absolute number of cells recovered on day 6 was higher in B-
2S cells than B-1P cells due to their increased proliferation
(Supplementary Fig. S1).
B-1P cell response to other TLR ligands was tested to determine
if B-1P hyporesponsiveness was restricted to TLR4 ligand. B-1P
cells proliferate significantly less than B-2S cells when stimulated
with various TLR agonists, including Pam3CSK4 (TLR-1/2),
poly(I:C) (TLR3), loxoribine (TLR7) and CpG (TLR9). In parallel,
both B-1P and B-2S cells proliferated equally well when stimulated
via CD40 (Fig. 1B). The TLR5 agonist, flagellin, failed to stimulate
B-1P and B-2S cell proliferation. Real time RT-PCR analysis
showed that TLR3, TLR4 and TLR7 mRNAs were expressed
equally in both B-1P and B-2S subsets, while TLR2 and TLR5
genes were expressed less in B-1P than in B-2S cells. Interestingly,
TLR9 expression was 3 fold higher in B-1P cells than in B-2S cells
(Fig. 1C). Therefore, the hyporesponsivness of B-1P cells to TLR3,
4, 7 and 9 ligands is unlikely to be due to decreased expression of
TLR on B-1P cells. We chose LPS and CpG for further studies
since LPS uses both TRIF and MyD88 pathways while CpG uses
only the MyD88 pathway. They also represent TLRs that differ in
cellular expression.
Hyporesponsiveness of B-1P cells to TLR signaling is in
part due to increased production of IL-10
An examination of the time kinetics revealed that B-1P and B-
2S cell proliferation responses to LPS and CpG were similar after
24 hours of stimulation. However, B-2S response peaked at
48 hours and remained significantly higher than B-1P cells up to
72 hours (Fig. 2A). This difference in kinetics, which is also
dependent on cell densities, may be the reason why many previous
studies [19,30,31,32] did not appreciate the difference in TLR
response of B-1 and B-2 cells. IL-10 inhibits in vitro proliferation of
murine B-2S cells and human activated leukemic CD5+B cells
[33,34]. Hence, we hypothesized that hyporesponsiveness of B-1P
cells to LPS and CpG may be due to production of IL-10 by B-1
cells and its inhibitory effects. However, the kinetics of IL-10
production by B-1P and B-2S cells upon TLR stimulation has
never been examined previously. As shown in Fig. 2B, B-1P cells
produced large amounts of IL-10, with quantities detectable as
early as 6 hours after LPS or CpG stimulation, while B-2S cells
produced very low levels of IL-10, which became detectable only
after 12 hours of LPS (TLR4) or CpG (TLR9) stimulation. At
48 hours, B-1P cells produced 10 fold more IL-10 upon LPS
stimulation and 25 fold more IL-10 upon CpG stimulation than B-
2S cells (Fig. 2B). Upon neutralization of IL-10 effects with anti-
IL-10R antibody, there was a significant (p,0.0004) increase in
LPS and CpG induced proliferation of B-1P cells at 48, 72 and
96 hours, but not at 24 hours (Fig. 2C). There was no significant
difference (p.0.05) in B-2S cell proliferation with anti-IL-10R
antibody treatment (Supplementary Fig. S2). Next, we examined if
B-1 cell derived IL-10 also plays a role in their differentiation
responses. Neutralization of IL-10 increased antibody production
by TLR4 or TLR9 stimulated B-1P cells (Fig. 2D), but no
significant difference was observed in the B-2S group with the
same treatment (Supplementary Fig. S3).
Agonists for TLR2, 3 and 7 also behave like TLR4 and TLR9
agonists in inducing very high amounts of IL-10 secretion by B-1P
cells but not by B-2S cells (Table 1). In terms of absolute
magnitude, IL-10 induced by TLR ligands ranged from 4 to
12 ng/ml for B-1P cells whereas it was 0.01 to 0.13 ng/ml for B-
2S cells. It is also interesting to note that B-1P cells produced high
Figure 1. B-1P cells respond weakly to TLR stimulation compared to B-2S cells. (A) B-2S and B-1P cells from C57BL/6 mice were cultured
with anti-IgM F(ab’)2, LPS-2880 (phenol extracted), LPS-2663 (gel purified), LPS-4005 (TCA precipitated) or anti-CD40 for 48 hours and proliferation
was measured by
independent experiments. (B) B-1P and B-2S cells were cultured with TLR2, 3 and 7 ligands Pam3CSK4, poly I:C, loxoribine, CpG respectively or anti-
CD40 for 48 hours and proliferation was measured by3[H] thymidine incorporation. Similar results were obtained in two other experiments. Data
points represent mean 6 SD values from triplicate cultures. In panels A and B, *** =p,0.0005 when comparing responses of B-1 and B-2S cells (C) B-
1P and B-2S cells were purified and rested for 30 minutes. Total RNA was isolated, and mRNA for various TLRs (TLR-2, 3, 4, 5, 7 and 9) and GAPDH were
quantified by quantitative real-time PCR as described under ‘‘Experimental Procedures’’. Amount of TLR mRNA in B-1P cells was expressed as a
percentage of corresponding TLR mRNA in B-2S cells. Data are presented as mean 6 SD values from triplicates and are representative of two
independent experiments. In all the panels 6-8 mice were used in each experiment.
doi:10.1371/journal.pone.0011445.g001
3[H] thymidine incorporation. Data are presented as mean 6 SD from triplicate cultures and are representative of three
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levels of IL-10 constitutively (Table 1). IL-10 appeared to have a
role in the reduced B-1 cell responses to all the TLR ligands
studied, since there was a significant (p,0.006) increase in
proliferation of B-1P cells when stimulated with different TLR
agonists in the presence of anti-IL-10R antibody in comparison to
stimulation with TLR agonists alone (Table 1).
B-1a cells, among different B cell subsets, produce high
amounts of IL-10 constitutively and upon TLR stimulation
B-1P cells can be further divided into B-1a and B-1b cells, both
of which express Mac-1 and B220, but only B-1a cells express
CD5. It has been shown that CD5 promotes IL-10 production in
human B cells [10]. Furthermore, CD5+B lymphoma cells
produce increased levels of IL-10 relative to CD52lymphoma
cells [35]. We purified the three peritoneal B cell subsets, B-1a
(B220+Mac-1+CD5+),B-1b (B220+Mac-1+CD52)
(B220+Mac-12CD52) cells, by FACS sorting (Fig. 3A). Among
these highly pure subsets (.98%), B-1a cells produced constitu-
tively higher amounts of IL-10 (161 pg/ml) compared to B-1b
(32 pg/ml) and B-2P (3 pg/ml) cells. B-1a cells also produced very
high levels of IL-10 upon LPS stimulation; almost 12 fold more
than B-2P cells and 3.5 fold more than B-1b cells (Fig. 3A, bottom
panel). The high amounts of IL-10 produced by B-1a cells
inhibited their own proliferation, while the amounts of IL-10
produced by B-1b and B-2P cells (Supplementary Fig. S4) were
not sufficient to inhibit their own proliferation. We also FACS-
purified the different splenic B cell subsets, specifically the
follicular and marginal zone B cells and tested for their ability to
produce IL-10 constitutively and after LPS stimulation. As shown
in table 2, none of the splenic B cell subsets produced constitutively
and B-2P
high levels of IL-10. Similarly, none of the splenic B cell subsets or
lymph node B cells produced high amounts of IL-10 upon LPS
stimulation in comparison to peritoneal B-1a cells. Although
marginal zone B cells produced as much IL-10 as B-1b cells, like
the B-1b cells the amount of IL-10 produced was not sufficient to
inhibit their own proliferation (Supplementary Fig. S5).
B-1S cells behave more like B-2S cells, rather than B-1P
cells, in terms of IL-10 production and proliferation
Even though the majority of B-1 cells are in the peritoneum,
substantial numbers of B-1 cells are also located in the spleen. B-
1P cells, unlike splenic B-1 (B-1S) cells, express a constitutively
activated form of STAT3, a transcription factor, known to
regulate IL-10 gene expression [30]. B-1S cells from VH12
transgenic mice (VH12 Tg) (Most VH12 B cells in adult mice bind
the common phospholipid phosphatidylcholine (PtC) and are B-1
[36]) showed very low constitutive IL-10 production and upon
LPS or CpG stimulation in comparison to B-1P cells (Fig. 3B, left
panel). Furthermore, B-1S cells from VH12 Tg mice did not show
any hyporesponsiveness to LPS or CpG stimulation in comparison
to B-2S cells from VH12 wild type littermate (VH12 WT) (Fig. 3B,
right panel). We could not detect any IL-10 in unstimulated wild
type B-1S cells even after FACS sorting (Fig. 3C, top panel),
suggesting that both B-2S and B-1S cells lacked the ability to
produce IL-10 constitutively. Upon LPS stimulation, both B-1S
and B-2S produced comparable amounts of IL-10 (Fig. 3Cbottom
left panel, ), but the amount is significantly less than B-1P cells
(Fig. 2B & 3A). As expected, the low levels of IL-10 produced by B-
1S and B-2S cells did not have an inhibitory effect on their
proliferation response to LPS, as shown by a lack of increase in
proliferation upon IL-10 neutralization (Fig. 3C, bottom right
panel). This data regarding IL-10 production by splenic B-1a cells
is in agreement with the identification of IL-10 producing B10
cells, characterized as CD1dhighCD5+cells, by Yanaba et al. [6].
However, it is unclear if the slpenic CD1dhighCD5+B10 subset will
produce as much IL-10 as the peritoneal B-1a cells, since Yanaba
et al. [6] used only intracellular staining to identify B-10 cells.
IL-10 regulates B-1P response: in vitro and in vivo
Since neutralization of IL-10 signaling via the anti-IL-10R
antibody led to an increased response of B-1P to TLR stimulation,
we determined if the B-1P cells from IL-102/2mice were also more
responsive to TLR signaling. B-1P cells from young IL-102/2mice
proliferated significantly more upon LPS (.5 fold) and CpG (.3
fold) stimulation when compared to B-1P cells from wild type mice
(Fig. 4A). Wild-type and IL-102/2B-1P cells proliferated equally
upon CD40 stimulation. The TLR responses of IL-102/2B-1P cells
were still susceptible to IL-10 inhibition, since exogenously added IL-
10 inhibited their response to LPS and CpG (Fig. 4A). LPS (data not
shown) orCpG(Fig.4B)induced B-1P (WTand IL-102/2) and B-2S
proliferation was inhibited similarly by exogenous IL-10.
Figure 2. Hyporesponsiveness of B-1P cells to different TLRs is due to high IL-10 production. (A) B-1P and B-2S cells were stimulated with
LPS or CpG for 24, 48, 72 and 96 hours in the presence or absence of anti-IL-10R antibody. The cultures were pulsed with3[H] thymidine during the
final four hours of the culture period and the results are presented as mean 6 SD of triplicate determinations. (B) Culture supernatants of B-2S and B-
1P cells were collected at 6, 12, 24 and 48 hours of stimulation with LPS or CpG and assayed by ELISA for IL-10. In panels A and B the p-values
(*=p,0.05, **=p,0.005, ***=p,0.0005) denote the significance of differences between the responses of B-1P and B-2S cells. Results presented in
panels A and B are representative of two independent experiments with 6–8 mice in each experiment. (C) B-1P cells were cultured with LPS or CpG in
the presence or absence of anti-IL-10R antibody or isotype antibody and proliferation was measured by3[H] thymidine incorporation. Data (mean 6
SD, n=10 mice per experiment) are representative of three independent experiments. The p-values (*=p,0.05, ***=p,0.0005) signify the
differences between proliferation responses with and without anti-IL-10 antibody. (D) B-1P cells were cultured with LPS or CpG for 5 days in the
presence or absence of anti-IL-10R antibody. At the end of 5 days, culture supernatants were collected and assayed by ELISA for total IgM. Results
(mean 6 SD, n=6 mice per experiment) are representative of three experiments. The p-value (***=p,0.0005) signify differences in antibody
secretion with and without anti-IL-10 antibody.
doi:10.1371/journal.pone.0011445.g002
Table 1. TLR induced IL-10 production by B-2S and B-1P cells
and its inhibitory effects on B-1P cells.
B-2S B-1P
TLR Ligand
IL-10
(pg/ml)
IL-10
(pg/ml)
% increase in
proliferation with
anti-IL-10R Ab
p-value
Media3 345--
TLR2Pam3CSK4 434187 1690.0001
TLR3Poly I:C 158019 166 0.0013
TLR7 Loxoribine12911990 154 0.0100
B cell subsets were cultured at 105cells/well with or without different TLR
ligand for 2 days. IL-10 was quantified by ELISA. Proliferation was measured
after 48 hours either in the presence or absence of a-IL-10R Ab (1 mg/ml). The
tritiated thymidine incorporation for B-1P cells stimulated with Pam3CSK4, Poly
I:C and Loxoribine in the absence of a-IL-10R Ab was 9830, 6989 and 9677,
respectively.
doi:10.1371/journal.pone.0011445.t001
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Since there was no difference in proliferation response of B-1
and B-2 cells at 24 hours (Fig. 2A), nor was the 24 hour response
enhanced by IL-10 neutralization in B-1P cells (Fig. 2C), we
hypothesized that the TLR induced IL-10 mediated inhibition
does not affect the first or initial rounds of cell division, as it takes
time to accumulate high levels of IL-10. CFSE staining revealed
that wild-type B-1P cells have decreased number of cells in the
second and subsequent rounds of cell division when compared to
IL-102/2B-1P cells upon LPS or CpG stimulation (Fig. 4C),
which supports our hypothesis. A cell cycle analysis by Propidium
Iodide (PI) staining showed that upon LPS stimulation, fewer wild-
type B-1P cells were present in S/G2/M phase when compared to
wild-type B-1P cells stimulated in the presence of anti-IL-10R
antibody or in IL-102/2B-1P cells (Fig. 4D). No significant
difference was observed in the B-2S group with the same
treatment (Supplementary Fig. S6). Thus, IL-10 appears to arrest
B-1 cell division at the G0/G1 stage of the cell cycle.
We further analyzed the IL-10 auto-regulatory effect on B-1P cells
in vivo. To demonstrate the autocrine and paracrine effects of B-1 cell
derived IL-10, we transferred 36106CFSE-labeled peritoneal B cells
from wild type and IL-102/2mice into Rag-12/2mice intraperi-
toneally. The recipients were challenged with 25 mg/ml LPS. The
CFSE-labeled B cells were then isolated from the peritoneal cavity
and the CFSE fluorescence of B-1a and B-1b B cellswas analyzed.As
shown in Fig. 4E total peritoneal B cells as well as B-1a cells from IL-
102/2mice exhibited greater proliferation than same subsets from
the wild type mice suggesting that IL-10 produced by B-1a cells can
have autocrine inhibitory effects in an in vivo situation. Moreover, B-
1b cells from IL-102/2mice also exhibited increased proliferation
suggestingthatIL-10fromB-1acells(and/orbythemselves)canhave
paracrine inhibitory effects.
Alternate NF-kB signaling, but not JAK/STAT signaling,
can overcome inhibitory effects of IL-10 on B-1P cells
Although CD40 stimulation induced IL-10 production (950 pg/
ml) by B-1P cells, the levels were much lower in comparison to
TLR4 stimulation (5380 pg/ml). No noticeable differences were
found in CD40 induced proliferation of B-1P and B-2S cells
(Fig. 1A & Fig. 1B). Moreover, neutralization of IL-10 signals with
anti-IL-10R antibody did not change the proliferation response of
either B-1P or B-2S cells to anti-CD40 (Fig. 5A). Hence, we
examined if CD40 co-stimulation can overcome the IL-10
mediated inhibition of TLR responses in B-1P cells.
CD40 co-stimulation with LPS resulted in a proliferation
response that was more than additive, which was not further
enhanced upon neutralization of IL-10 signals, but was reduced
slightly (Fig. 5B). CD40 signals via both alternate and classical NF-
kB pathways, whereas TLRs signal via the classical NF-kB
pathway [37,38]. In monocytes, exogenous IL-10 inhibits TLR
signaling by blocking classical NF-kB pathways via inhibition of
IkB kinase (IKK) activity or p50/p65 translocation to nucleus, or
by selectively inducing nuclear translocation of p50/p50 homo-
dimer [39,40,41]. Hence, absence of inhibition of CD40 responses
could be due to the activation of alternate NF-kB pathway. BAFF,
which is critical for survival and maturation of B cells, also signals
via the alternate NF-kB pathway [38]. Therefore, we examined if
BAFF co-stimulation can overcome the IL-10 mediated inhibition
in B-1 cells. BAFF co-stimulation with LPS leads to a synergistic
proliferation response, which was not enhanced by IL-10
neutralization (Fig. 5C). Instead we found a slight decrease in
proliferation response of B-1P cells upon LPS co-stimulation with
anti-CD40 or BAFF in the presence of anti-IL-10R antibody. It is
conceivable that in the presence of CD40 and BAFF, two agents
Figure 3. B-1a cells are the major IL-10 producing cells among peritoneal and splenic B cell populations. (A) Macrophage depleted
peritoneal cell populations were gated on B220+cells and further separated into B-1a (Mac1+CD5+), B-1b (Mac1+CD52) and B-2 (Mac12CD52) using a
FACS machine (top panel). Sorted B-1a, B-1b and B-2 cells were cultured with LPS for 48 hours. The culture supernatants were collected and assayed
for IL-10 (bottom panel). Data are shown as mean 6 SD of triplicate cultures and are representative of two independent experiments with 15 mice
each. The p-values signify the differences in IL-10 production by B-1a cells in comparison to B-1b and B-2P cells (***=p,0.0005) and between B-1b
and B-2P cells (**=p,0.005). (B) B-1P cells from C57BL/6 and B-1S cell from VH12 Tg mice were cultured with LPS or CpG for 48 hours; culture
supernatants were collected at 48 hours and assayed for IL-10 (left panel). B-2S cells from VH12 wild type littermate (WT) mice and B-1S cell from VH12
Tg mice were cultured with LPS or CpG for 48 hours and cell proliferation was assessed by3[H] thymidine incorporation (right panel). Similar results
were obtained in two other experiments (mean 6 SD with five mice per group). The p-value (***=p,0.0005) signify the differences in response
between B-1P and B-1S cells. (C) Total spleen cells from C57BL/6 mice were sorted into B-2S (B220+CD52) and B-1S (B220+CD5+) cells on the basis of
CD5 expression. Sorted B-2S and B-1S cells were cultured with LPS for 48 hours, culture supernatants were collected and assayed for IL-10 (bottom
left panel), and for proliferation (bottom right panel) with or without anti-IL-10R antibody. Representative results from the one of two experiments
are shown (mean 6 SD, n=5).
doi:10.1371/journal.pone.0011445.g003
Table 2. Constitutive and LPS induced IL-10 production by various murine B cell subsets.
Anatomical location B cell subset Surface phenotype
IL-10 (pg/ml)
Media LPS
PeritoneumB-1a cells B220+Mac-1+CD5+
B220+Mac-1+CD52
B220+Mac-12CD52
B220+Mac-12CD5+
B220+AA4.12CD21hiCD23lo
B220+AA4.12CD21intCD23hi
B220+
B220+
16163240636406
B-1b cells
326911936126
B-2 cells
361 3376166
SpleenB-1 cells
ND583642
Marginal Zone B cells
965 1054632
Follicular B cells
162136612
Total B cells
ND 313626
Mesenteric Lymph NodesTotal B cells
162240618
B cell subsets in the peritoneum, spleen and mesenteric lymph node were separated by FACS sorting ($96% pure) and were cultured at 105cells/well with or without
5 mg/ml LPS for 2 days. IL-10 was quantified by ELISA. ND = non-detectable.
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known to stimulate alternate NF-kB signaling, IL-10 has a
prosurvival effect on B cells.
B-1 cells express the IL-5 receptor and IL-5 acts as a survival
and proliferation factor for B-1 cells by signaling via JAK/STAT
pathway [42]. IL-5 co-stimulation with LPS resulted in an increase
in proliferation response which was significantly enhanced when
IL-10 signaling was blocked (Fig. 5D). Similar results were
obtained with IL-2 and IL-4 (data not shown). Thus, co-
stimulation via alternate NF-kB signaling (CD40, BAFF), but
not JAK/STAT signaling (IL-2, IL-4, IL-5), can overcome the IL-
10 mediated inhibition of B-1P cells upon TLR stimulation.
IL-10 inhibits NF-kB nuclear translocation by preventing
LPS-induced degradation of IkBa
Since IL-10 inhibited LPS (known to activate NF-kB by the
classical pathway) but not CD40 (known to activate NF-kB by
both classical and non-classical pathways) induced B-cell growth
responses, we tested if IL-10 has differential effects on the two
pathways of NF-kB activation. We used B-2 cells for these studies
because both B-2 and B-1 cells are similarly susceptible to IL-10
mediated inhibition when stimulated with TLRs (Fig. 4B). B-2S
cells were stimulated with LPS for indicated time points in the
presence or absence of IL-10 (as described in Methods). As shown
in Fig. 6A, LPS stimulation resulted in a time-dependent
degradation of IkBa. In contrast, pretreatment of IL-10 decreased
IkBa degradation. To further elucidate if IL-10 induced blocking
of IkBa degradation inhibits RelA (p65) translocation into nucleus,
we examined the accumulation of RelA in the nuclear fraction of
LPS stimulated cells in the presence or absence of IL-10. LPS
stimulated cells showed increased nuclear translocation of RelA
compared to IL-10 pretreated cells (Fig. 6B). There was up to a 9
fold increase in nuclear translocation of RelA upon LPS
stimulation in the absence of IL-10. However, there was only a
2.5 fold increase in nuclear translocation of RelA when cells were
pre-incubated with IL-10 (Fig. 6B).
As noted above, CD40 signaling can activate NF-kB by the
alternate pathway, which involves translocation of RelB into the
nucleus. We confirmed this by showing that RelB is found in
increased amounts in the nuclear lysates of CD40-activated B cells
(Fig. 6C). Treatment with IL-10 did not affect nuclear levels of
RelB in CD40 stimulated B cells at most time points examined. At
two time points (309 and 459) there was a small decrease in nuclear
levels of RelB in IL-10 treated cells which was not sustained at the
609 time point. These results all together indicate that pretreate-
ment with IL-10 blocks LPS induced classical NF-kB pathway, via
IkBa degradation and RelA translocation to nucleus, but not the
CD40 induced alternate NF-kB pathway.
Production of IL-10 by B-1P cells limits clearance of B.
hermsii from the blood
B-1P cells play a primary role in the clearance of B. hermsii [12].
TLR2 plays a major role in the activation of B cells when they
come in contact with B. hermsii lipoproteins and leads to B cell
proliferation and differentiation to produce antibodies against B.
hermsii lipoproteins [43]. Even though, TLR2 signaling plays a
major role in the activation of B-1 cells, it is not the only TLR
required for response against B. hermsii, as MyD88 knockout mice
suffer from more severe episodes of bacteremia with B. hermsii than
TLR2 knockout mice [28].
Accordingly, we found that B-1P cells produce high levels of IL-
10 when stimulated with B. hermsii (Fig. 7A), as we have shown
previously with a synthetic TLR2 ligand (Table 1). We also found
that B. hermsii induced greater proliferation of wild type B-1P cells
upon blocking of IL-10 signaling with a-IL-10R antibody (Fig. 7B,
left panel). Similarly, B-1P cells from the IL-102/2mice
proliferated better than wild type mice, presumably via a B.
hermsii associated TLR2 (Fig. 7B, right panel). There is also an
increase in antibody production by IL-102/2B-1P cells compared
to WT B-1P cells upon B. hermsii stimulation (Fig. 7C).
Therefore, the ability of B-1 cells to produce antibody and clear
the B. hermsii infection might become restricted due to high levels
of IL-10 produced by B-1 cells. To test this hypothesis, we
adoptively transferred purified B-1P cells from wild-type or IL-
102/2mice into cohorts of mMT mice (B cell deficient mice). The
mMT mice cannot eliminate B. hermsii from their blood [44]. The
control untransferred mMT mice maintained 1786103bacteria/ml
of blood up to 10 days without any relapse. The B-1 cells provided
by adoptive transfer were sufficient to control B. hermsii, as both
groups of adoptively-transferred mice achieved peak bacteremia at
day 2 and cleared bacteria by day 5 or 6. Interestingly, levels of B.
hermsii in the blood of mice which received wild-type B-1P cells
were significantly higher than in the mice that received IL-102/2
B-1P cells (days 2 to 4, Fig. 7D). The first bacteremic episode
peaked at bacterial densities that were nearly twice as high in mice
that received wild-type B-1P than in mice that received IL-102/2
B-1P cells (p,0.0005; Fig. 7D). Although there was some mouse to
mouse variation, there were no significant differences in the
persistence of the first bacteremia episode of either group. The
second bacteremia peak also trended toward higher bacterial
densities in the wild-type B-1 cell transferred group than in the IL-
102/2B-1 cell transferred group, but the differences were not
statistically significant (p,0.11; Fig.7D). These results indicate that
B-1 cell derived IL-10 plays a significant role during B. hermsii
infection and impairs the ability of B-1 cells to clear B. hermsii from
the blood.
Figure4.IL-10inhibitsB-1PcellresponsebyarrestingthecellsinG0/G1phaseofcellcycle.(A)B-1PcellsfromC57BL/6andIL-102/2micewere
cultured with LPS, CpG or anti-CD40 for 48 hours. IL-10 (2500 pg/ml) was added to some of the cultures of IL-102/2B-1P cells. Results (mean 6 SD of
triplicate cultures) are representative of three experiments with seven mice per experiment. *=p,0.05 indicates statistical significance of the response
between WT B-1P and IL-102/2B-1P or also with and without added IL-10. (B) Dose dependence of IL-10 mediated inhibition of B-2S,WT B-1P and IL102/2
B-1Pcell proliferation responses to CpG. Similar resultswere obtained in three other experiments with4–6 miceper experiment.(C) CFSE labeled B-1P cells
(105/well)fromC57BL/6andIL-102/2micewereculturedwithLPSorCpGfor48 hoursandwereanalyzedforCFSEdilutionaftergatingforB220andCD11b
double positive cells. The percentage cells in M1 regional gate represent CFSE dilution from the second and subsequent divisions of cells based on the
modfit analysis of CFSEfluorescence(D)B-1P cells from C57BL/6 and IL-102/2micewere cultured with LPS or anti-CD40in thepresenceor absence of anti-
IL-10Rantibodyfor48 hoursandcellcycleanalysiswasperformedbyPIstaining.ThefractionofcellsinG2/S/Mwasdeterminedfromtriplicateculturesand
the mean 6 SD values are plotted. Similar results were obtained in two other experiments with seven mice per group. The statistical significance of
difference in response with or without anti-IL-10R antibody or between wild type and IL-10 knockout B-1P cells is shown by *=p,0.005. (E) CFSE labeled
purified peritoneal B cells (B220+) from wild type or IL-102/2mice were injected intraperitoneally into Rag-12/2mice. Two hours later 25 mg of LPS per
mousewasinjectedintraperitoneally.TotalB-1Pcells (B220+) (left panel),B-1acells(B220+Mac-1hi) (middlepanel) andB-1bcells(B220+Mac-1lo)(rightpanel)
fromwildtypeor IL-102/2transferredmicewereanalyzedfor CFSE dilutionafter60 Hours ofinvivostimulation withPBSor LPS.(*=p,0.005,comparedto
PBS treated group, **=p,0.005, compared to WT B-1 transferred group with LPS stimulation)
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Discussion
Studies presented here identified the novel property of autoreg-
ulation in the B-1 B cell subset, wherein IL-10 produced by B-1 cells
inhibits their own functional responses. We have demonstrated that
the TLR induced response of B-1P cells is less than that of B-2S cells
due to rapid induction of IL-10, which in turn suppresses the
proliferation and differentiation of B-1P cells. We used both the anti-
IL-10R antibody and IL-102/2B-1P cells to demonstrate the
inhibitory effects of IL-10 on TLR responses of B-1P cells. The
susceptibility to inhibition by IL-10 was not unique to the B-1 subset,
as similar effects were observed in all studied B cell subsets once
adequate levels of IL-10 were present. B-1P cells were unique in that
they produced very high levels of IL-10 leading to a feedback
inhibition of their TLR responses and hence we call them
‘‘autoregulatory B cells’’. This autoregulatory property of B-1P cells
appears to have a physiological affect in moderating the B-1 cell
responses to bacterial infection. The inhibitory effect of high doses of
IL-10 on B cell proliferation is consistent with previously reported
observations that high levels of exogenous IL-10 inhibit proliferation
of human leukemic CD5+B-cells [33], superantigen induced T cells
[45] and macrophage proliferation [46]. The IL-10 mediated
autoregulation is not restricted to TLR responses, since similar
effects were also observed in B-1P cell response to BCR cross-linking
(manuscript in preparation).
The rapid and high levels of IL-10 production were observed
with all TLR ligands studied except flagellin, a TLR5 ligand,
which did not induce a measurable proliferation response. The B-
1 cell proliferation response to all the TLR ligands was less than
that of the B-2 cell response and in all cases could be enhanced by
neutralization of the inhibitory effects of IL-10. Thus, the IL-10
induction by TLR ligands was seen with both MyD88 dependent
and independent stimuli. This may relate to the previously
described constitutive expression of STAT-3 by B-1 cells, a
transcription factor required for IL-10 production [30]. The
transcription factors Sp3, c-maf, IRF, CREB and NF-kB have a
role in IL-10 transcription [47]. Moreover, IL-10 has also been
shown to be regulated at the level of mRNA stability and
translation. Future studies will have to determine if any of these
factors are also upregulated in the B-1P cell subset to account for
their high IL-10 phenotype. Preliminary studies suggest that
activated p38MAPkinase may have some role in this, since
p38MAPK inhibitors partially mimic the anti-IL-10R antibody
treatment in enhancing B-1P cell functional response (Sindhava
et al. unpublished data).
The production of high levels of IL-10 both constitutively and
after TLR signaling was uniquely associated with the B-1a cell
subset and to some extent with the B-1b B cell subset in the
peritoneum. B-1a cells showed constitutive IL-10 production, 53
fold higher than peritoneal B-2 and 5 fold higher than peritoneal
B-1b cells. Upon LPS stimulation, B-1a cells showed the highest
IL-10 production among the peritoneal B cell subsets. This high
IL-10 production by B-1a cells also inhibits their own
proliferation in an autocrine manner, whereas lower levels of
IL-10 secretion by B-1b cells is not enough to inhibit their own
proliferation upon LPS stimulation. However, in vivo, the high
levels of IL-10 produced by B-1a cells in the peritoneal cavity are
capable of suppressing the B-1b B cell responses as demonstrated
by our adoptive transfer experiment (Fig. 4E). Thus the IL-10
produced by B-1a cells can have both autocrine and local
paracrine effects.
Initially, VH12 Tg mice were used as a source of B-1 cells from
the spleen. B-1S cells from the mice produced 8-fold less IL-10 in
comparison to B-1P cells. Sort-purified B-1S (B220+CD5+) cells
from wild-type mice spleens also behaved like VH12 Tg B-1S cells.
Accordingly, there was no significant difference in proliferation
between VH12 Tg B-1S, wild type B-1S and wild type littermate
B-2S cells upon LPS stimulation. Additionally, the proliferation of
B-1S cells is not affected by the low levels of IL-10 secreted by
these cells. Together these results demonstrate that B-1S cells
behave more like a B-2S cell, rather than B-1P cells, in terms of IL-
10 production and proliferation.
These results do not contradict the identification of IL-10
producing B10 cells in spleen by Yanaba et al. [6]. We do find IL-
10 production by Splenic B-1 cells but quantitatively it is less than
that induced by peritoneal B-1a cells. Since the B-1S cells from
wild-type mice or VH12 Tg mice do not exhibit high IL-10
secretion, it is likely that the peritoneal environment has a unique
role in the ability of B-1P cells to produce IL-10. Previously,
Chumely et al. as well as Stoermann et al. have described the
existence of factors in the peritoneum that affect the special
characteristics of B-1P cells [48,49]. The above described
constitutive STAT-3 expression may also have a role in IL-10
production by B-1P cells, although STAT-3 expression has been
found to be an intrinsic but not an induced property of B-1P cells
[30]. Since B1P cells have a major role in mucosal IgA production,
it will be interesting to determine if B cells from the mucosal sites
also share the property of high IL-10 production with B-1P cells
and how they escape the inhibitory effects of IL-10 to produce
large quantities of IgA.
Among B cell subsets, marginal zone B cells and transitional 2 B
cells, as well as the recently described splenic B10 cell subset, have
all been reported to produce IL-10 [5,6]. The B10 subset expresses
CD5 and high levels of CD1d, but CD1d expression is not
required for IL-10 production as B-1P cells do not express CD1d
[50]. Most of the previous studies used RT-PCR or intracytoplas-
mic staining, techniques which do not allow for accurate
quantification of the secreted IL-10 and thus were unable to
appreciate the significant differences between the levels of IL-10
produced by the various B cell subsets. In a side by side
comparison, our studies found that the peritoneal B-1a subset
produced more IL-10 than follicular B cells, marginal zone B cells
and B-1S cells. Not only do B-1P cells produce high levels of IL-
10, but they also exhibit accelerated kinetics, which allows for
rapid accumulation of IL-10 in cultures and leading to inhibition
of the TLR response.
The effect of IL-10 is mainly on the activation of NF-kB by the
classical pathway, since IL-10 inhibited the nuclear translocation
of LPS-induced p65 but not of CD40-induced p52. This explains
why stimuli such as CD40 and BAFF that can signal via the
alternate NF-kB pathway are able to overcome the inhibitory
effects of IL-10 on TLR responses. Future studies will determine if
IL-10 inhibits the upstream NIK, but not the MEKK3, that are
pivotal points that distinguish classical and alternate pathways of
NF-kB activation.
Figure 5. CD40 and BAFF, but not IL-5, overcome the inhibitory effects of IL-10. B-2S and B-1P cells were cultured with (A) a-CD40 (B) LPS,
anti-CD40 or both together (C) LPS, BAFF or both together (D) LPS, IL-5 (10 ng/ml) or both together for 48 hours in the presence or absence of anti-
IL-10R antibody. Cell proliferation was measured by3[H] thymidine incorporation. Results (shown as mean 6 SD responses of triplicate cultures) are
representative of three experiments with 8 mice per experiment. The p-values (*=p,0.05, **=p,0.005, ***=p,0.0005) depict significance of
difference in the proliferation response with and without anti-IL-10R antibody.
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Figure 6. Effect of IL-10 on LPS and CD40 induced NF-kB activation in B-2S cells. Lysates from B-2 S cells at different time points were
analyzed by western blot technique as described under ‘‘materials and methods.’’ (A) IkBa protein expression in cytoplasmic fractions of LPS-
stimulated cells. (B) RelA protein expression in nuclear fractions of LPS-stimulated cells. (C) RelB protein expression in nuclear fractions of CD40-
stimulated cells. b-actin and lamin A\C were used as a control for cytoplasmic and nuclear fractions, respectively. Fold difference in the protein
expression are represented in line graphs after normalization with loading controls.
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Several studies have demonstrated that IL-10 can decrease the
antimicrobial activities of immune cells. There are also reports
that mice in which IL-10 affect has been neutralized show
enhanced abilities to clear pathogens such as Streptococcus, Listeria
and Mycobacterium species, when compared to mice with
functionally active IL-10 [51]. In many of these studies, IL-10
has been shown to affect several cell types, including CD4+T
cells and macrophages. Clearance of the relapsing fever causing
bacterium, B. hermsii, is dependent upon B-1P cells: mice
deficient in TLR1, TLR2, or the TLR adaptor protein
MyD88 generated anti-B. hermsii IgM with delayed kinetics and
suffered more severe episodes of bacteremia [28]. The present
study found that IL-102/2B-1P cells proliferated to significantly
higher levels than did wild-type B-1P cells upon B. hermsii
exposure in vitro, which is due to autoregulation by induced IL-10
production by wild type B-1P cells. Lazarus et al. observed that
IL-102/2mice are more effective at controlling B. burgdorferi, the
Lyme disease agent, which was attributed to a lack of IL-10
suppression of non-B-cells [52]. Here we showed that IL-102/2
B-1P cells were significantly better than wild-type B-1P cells in
controlling B. hermsii infection of mMT mice. Although the effects
of IL-10 on myeloid cells may also be important, we also have to
consider the direct impact of IL-10 on B cell production of
antibodies. In our adoptive transfer system, B-1P cells were the
only cells that differed in their ability to produce IL-10 as the
non B cells in the host were competent to produce IL-10. B-1b
cells have been shown to be more important than B-1a cells in
antibody production against Borrelia species [12]. Since we used
unseparated B-1 cells from wild-type and IL-10 deficient mice, it
is conceivable that in vivo B-1b cells also produce high levels of
IL-10. Alternatively, large quantities of IL-10 produced by
peritoneal B-1a cells inhibit anti-Borrelia antibody production by
the B-1b cells, which are also present in the peritoneal cavity, as
demonstrated by our Rag-12/2transfer experiment shown in
Fig. 4E. We will have to evaluate this phenomenon in infections
with other bacteria such as Streptococcus pneumoniae where B-1a
cells produce the innate antibody response and the B-1b cells
produce the adoptive antibody response [11].
B-1 cells have been known to secrete antibodies to single
stranded DNA, red blood cell surface molecules and several other
self antigens. In the rheumatoid factor transgenic mouse model,
Marshak-Rothstein and colleagues have shown that TLR9
signaling via DNA from dying cells is a critical factor for activation
of self-reactive B cells [29]. Also, BAFF and MyD88 signaling has
been shown to promote lupus like disease independent of T cells
[53]. Here we showed that BAFF can overcome IL-10 induced
down regulation of B-1 cell responses. Excess TLR activation as
seen in Yaa mice with TLR7 duplication or TLR7 transgenic mice
also leads to autoimmunity [54].
Conceivably, the autoregulatory properties of B-1 cells de-
scribed here may play a role in healthy individuals in preventing
excessive activation of self-reactive B cells via TLR stimulation.
Materials and Methods
Mice and reagents
C57BL/6 mice were obtained from Harlan (Indianapolis, IN,
USA). IL-102/2, Rag-12/2and mMT mice were obtained from
The Jackson Laboratory (Bar Harbor, ME, USA). VH12 Tg were
provided by Dr. Stephen Clarke (University of North Carolina,
Chapel Hill, NC) [36]. Mice were housed under specific pathogen-
free conditions in micro-isolator cages under the Institutional
Animal Care and Use Committee (IACUC) approved protocol.
The University of Kentucky IACUC protocol number for this
study is 00680M2004. The described studies are approved under
this protocol.
TLR agonists: LPS, CpG and Pam3CSK4 were obtained from
Sigma Chemical Co. (St. Louis, MO, USA), UCDNA (Calgary,
AB, Canada) and Calbiochem (San Diego, CA, USA), respective-
ly. Loxoribine, poly I:C and flagellin were obtained from
Invivogen (San Diego, CA, USA). The polyclonal goat anti-IgM
F(ab’)2 was obtained from ICN/MP Biomedicals, (Irvine, CA,
USA), and the anti-IL-10 receptor (anti-IL-10R) (Clone 1B1.3a)
was obtained from BD Biosciences (San Diego, CA, USA). Anti-
CD40 (1C10 clone) was a gift from Dr. Maureen Howard and was
used as ascites fluid. Anti-RelA, anti-IkBa and anti-b-actin
antibodies were obtained from Santa Cruz Biotechnology (Santa
Cruz, CA, USA), anti-RelB was obtained from Cell Signaling
(Danvers, MA, USA) and anti-lamin A\C was obtained from
Upstate (Temecula, CA, USA).
Purification of B cells
Peritoneal cells were obtained from 2–3 month old C57BL/6
mice by peritoneal lavage with Hank’s buffered salt solution.
Peritoneal and splenic B cells were purified as described in a
previous study [20]. Anti-B220/CD45RB,APC, anti-CD11b,
PE, anti-CD5,FITC, anti-CD21,FITC, anti-CD23,PE or anti-
AA4.1,APC (BD Pharmingen, San Jose, CA) were used to
identify and sort B-1a, B-1b, B-2, marginal zone B and follicular B
cells from the peritoneum or spleen of C57BL/6 mice using a
MoFlo cytometer (DakoCytomation) [55]. VH12 Tg mice and
wild-type C57BL/6 mice were used for splenic B-1 (B-1S) cell
purification.
In vitro cell proliferation assay and cytokine analysis
Splenic B-2 (B-2S), B-1S and\or B-1P cells (122.56105) were
stimulated with 50 mg/ml polyclonal goat anti-IgM F(ab’)2, 5 mg/
ml LPS, 5 mg/ml CpG, 5 mg/ml Pam3CSK4, 50 mg/ml poly I:C,
50 mg/ml loxoribine, 50 mg/ml flagellin or 1:1000 dilution of anti-
CD40 (1C10) ascites in the presence or absence of anti-IL-10R
antibody (1 mg/ml). Cultures were pulsed with3[H] thymidine for
4 hours on days 1, 2, 3 or 4, and then harvested (Packard,
Meriden, CT); the incorporated radioactivity was then measured
using a Matrix 96 b-counter (Packard, Downers Grove, IL).
Results are represented as mean 6 SD of triplicate cultures.
Figure 7. Role of IL-10 in B-1P cell responses to B. hermsii in vitro and in vivo. (A) B-1P cells from C57BL/6 mice were cultured with different
numbers of live B. hermsii or LPS for 48 hours and culture supernatants were analyzed for IL-10 secretion by ELISA. (B) Wild type B-1P cells in the
presence or absence of a-IL-10R antibody (left panel) or B-1P cells from C57BL/6 and IL-102/2mice (right panel) were cultured with different numbers
of live B. hermsii for 48 hours and proliferation was measured by3[H] thymidine incorporation. Representative results (mean 6 SD) from the one of
three experiments are shown (seven mice per group in each experiment). The statistical significance of difference in response with or without anti-IL-
10R antibody or between wild type and IL-10 knockout B-1P cells is shown by *=p,0.05. (C) B-1P cells from C57BL/6 and IL-102/2mice were
cultured with different numbers of live B. hermsii for 5 days and total IgM production was measured by ELISA. (D) mMT mice transferred with 26106
wild-type (n=4) or IL-102/2B-1P (n=5) cells were infected with B. hermsii and bacterial numbers in the blood were quantified by dark field
microscopy every 24 hours (left panel). Mean values of B. hermsii numbers in each group during the first and second peak (day-10) of bacteremia are
shown in the right panel. Results for the studies in panels B, C and D are from one of two experiments, both of which had similar outcome. For panel
C and D p-value (*=p,0.05) depict significance of difference in response between WT and IL-10 knockout B-1P cells.
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For cytokine analysis B-1P, B-1S and\or B-2S (122.56105) cells
were cultured in triplicate for indicated time points with various
stimulants. IL-10 levels in the supernatants were estimated using
ELISA with OptEIA kits (PharMingen, San Diego, CA, USA).
Results are presented as mean 6 SD of triplicate measurements
for triplicate cultures. Statistical significance of differences in
response was evaluated by unpaired Student’s t-test.
CFSE labeling and Cell Cycle Assays
B cells (resuspended at 107/ml in PBS/0.1% BSA, 10 mM
CFSE) were incubated at 37uC for 20 min, and then washed with
IF-12 medium +10% FBS as described by Lyons et al. [56]. CFSE-
labeled B cells (2.56105) were cultured in the presence of LPS
(5 mg/ml) or CpG (5 mg/ml) for 2 days at 37uC with 5% CO2.
B-1P and B-2S cells (2.56105) were cultured with 5 mg/ml LPS
or 1:1000 dilution of anti-CD40 for 2 days. Cells were fixed in
70% (v/v) ethanol for at least 1 h at 4uC, after which the cells were
incubated in a mixture of 1 mg/ml propidium iodine (PI) (Sigma-
Aldrich) and 25 mg/ml RNase A (Sigma-Aldrich) at 37uC for at
least 30 min. The level of PI fluorescence was measured with a
MoFlo flow cytometer. Cell populations at subG1, G1, S, G2/M
phase were calculated using the program ModFit.
Quantitative Real-time PCR
Total RNA was isolated from Purified B-1P and B-2S cells
(56106) with the RNA miniprep (Invitrogen, Carlsbad, CA). RNA
was quantified at OD260 using a Beckman DU 530 Life Science
UV Spectrophotometer (Beckman Coulter Inc, CA) and 2 mg of
total RNA was subsequently used to make cDNA using the
Superscript II reverse transcriptase (Invitrogen Corp., Carlsbad,
CA) according to the manufacturer’s protocol. RT-PCR was
performed on an ABI Prism 7000 system (Applied Biosystems,
Foster City, CA). Primers for murine TLRs were used as reported
previously [57]. The GAPDH-specific primers were used for
loading control (IDT technologies, Corallevielle, IA).
Peritoneal cell transfer and LPS administration
A total of 36106CFSE-labeled wild type or IL-102\2
peritoneal B cells were intraperitoneally injected into Rag-12/2
mice. Rag-12/2mice transferred with wild type peritoneal B cells
or IL-102\2peritoneal B cells were intraperitoneally injected with
either 250 ml of PBS (n=4) or 250 mL of LPS (100 mg/ml) (n=4)
2 hours after B cell-administration. Total peritoneal cells were
recovered from each mouse after 60 hours of PBS or LPS injection
and subjected to flow cytometry.
Western blot analysis
B-2S cells (106106), pre-incubated with or without IL-10 (5 ng/
ml, 12 hours), were stimulated with LPS (5 mg/ml) or anti-CD40
(1:1000) ascites for the indicated time. Cell pellets were lysed in a
nuclear and cytoplasmic extraction reagent (Thermo scientific),
proteins separated by SDS-PAGE and processed for western
blotting. The blots were developed with HyGLO chemilumines-
cence substrate (Denville scientific), exposed to Kodak X-Omat
films and analyzed by an Eastman Kodak Image Station 2000RT.
For re-probing, membranes were stripped using a solution
containing 62.5 mM Tris-HCl, 2% SDS, and 100 mM a ˆ-
mercaptoethanol at 50uC for 30 min.
Infection and quantification of B. hermsii in mice
B. hermsii isolate DAH was obtained from Dr. Tom Schwan
(Rocky Mountain Laboratories, NIH, Hamilton, MT, USA).
Efficient experimental B. hermsii infection is best achieved using
host-adapted spirochetes, i.e. bacteria taken directly from the
blood of other infected mice [44,58]. 8–12 week old C57BL/6
wild-type mice (‘‘donors’’) were infected by intraperitoneal
injection with 56105B. hermsii DAH from a mid-exponential
phase culture. At the peak of the first bacteremia, the donor mice
were euthanized and exsanguinated. Donor mouse blood in
citrate buffer was pooled and bacterial concentration determined
by darkfield microscopy using a Petroff-Hausser counting
chamber.
Two cohorts of B cell-deficient (mMT) mice were adoptively
transferred with B-1P cells (26106cells/mouse) purified from
either wild-type or IL-102/2mice. One week after transfer, the
mice were infected by intraperitoneal injection of 36105B. hermsii
harvested freshly from donor mice. mMT mice that had not
undergone adoptive transfer were likewise infected with B. hermsii,
as a control. Densities of B. hermsii in infected mouse blood were
quantified every 24 hours as described by Alugupalli et al. [12].
Statistical analysis
Paired student’s t-test was used to determine statistical
significance of differences between various groups.
Supporting Information
Figure S1
cells/well and stimulated with LPS (5 mg/ml). Cell viability was
measured at days -2, 4 and 6 by the trypan blue dye exclusion
method.
Found at: doi:10.1371/journal.pone.0011445.s001 (0.19 MB
TIF)
B2S and B1P were plated at a cell density of 10e5
Figure S2
(5 mg/ml) in the presence or absence of anti-IL-10R antibody
(1 mg/ml); proliferation was measured by 3[H] thymidine
incorporation.
Found at: doi:10.1371/journal.pone.0011445.s002 (0.14 MB
TIF)
B-2S cells (10e5 cells/well) were cultured with LPS
Figure S3
(5 mg/ml) or CpG (5 mg/ml) for 5 days in the presence or absence
of anti-IL-10R antibody (1 mg/ml). At the end of 5 days culture
supernatants were collected and assayed by ELISA for total IgM.
Found at: doi:10.1371/journal.pone.0011445.s003 (0.29 MB
TIF)
B-2S cells (10e5 cells/well) were cultured with LPS
Figure S4
(10e5 cells/well) were cultured with LPS (5 mg/ml) in the presence
or absence of anti-IL-10R antibody (1 mg/ml) for 48 hours. Cell
proliferation was determined by 3[H] thymidine incorporation.
Found at: doi:10.1371/journal.pone.0011445.s004 (0.32 MB
TIF)
FACS sorted peritoneal B-1a, B-1b and B-2P cells
Figure S5
follicular (Fo) B cells (10e5 cells/well) were cultured with LPS
(5 mg/ml) or a-CD40 in the presence or absence of anti-IL-10R
antibody (1 mg/ml) for 48 hours. Cell proliferation was deter-
mined by 3[H] thymidine incorporation.
Found at: doi:10.1371/journal.pone.0011445.s005 (0.65 MB
TIF)
FACS sorted splenic marginal zone (MZ) and
Figure S6
a-CD40 in the presence or absence of anti-IL-10R antibody
(1 mg/ml) for 48 hours and cell cycle analysis was performed by PI
staining.
Found at: doi:10.1371/journal.pone.0011445.s006 (0.24 MB
TIF)
B-2S cells (10e5 cells/well) were cultured with LPS or
B-1 Cell Autoregulation
PLoS ONE | www.plosone.org15 July 2010 | Volume 5 | Issue 7 | e11445