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Immunity
Article
Coactivation of Syk Kinase and MyD88 Adaptor
Protein Pathways by Bacteria Promotes
Regulatory Properties of Neutrophils
Xiaoming Zhang,1,2 Laleh Majlessi,1,2 Edith Deriaud,1,2 Claude Leclerc,1,2 and Richard Lo-Man1,2,*
1Institut
Pasteur, Unité Régulation Immunitaire et Vaccinologie, 25 rue du Dr. Roux, 75724 Paris Cedex 15, France
U883, 75724 Paris, France
*Correspondence: [email protected]
DOI 10.1016/j.immuni.2009.09.016
2Inserm,
SUMMARY
Neutrophils are one of the first lines of defense
against microbial pathogens and are rapidly recruited
at the infection site upon inflammatory conditions.
We show here that after bacterial stimulation, and in
contrast to monocytes and macrophages, murine
neutrophils contributed poorly to inflammatory responses; however, they secreted high amounts of
the anti-inflammatory cytokine IL-10 in a DAP12
adaptor-Syk kinase and MyD88 adaptor-dependent
manner. Cotriggering of TLR-MyD88- and C-type lectin receptor (CLR)-Syk-dependent pathways led to
a quick and sustained phosphorylation of p38 MAP
and Akt kinases in neutrophils. In vivo, both Gramnegative bacteria and mycobacteria induced the
recruitment of neutrophils secreting IL-10. In acute
mycobacterial infection, neutrophil-derived IL-10
controlled the inflammatory response of dendritic
cells, monocytes and macrophages in the lung.
During a chronic infection, neutrophil depletion
promoted inflammation and decreased the mycobacterial burden. Therefore, neutrophils can have a previously unsuspected regulatory role during acute and
chronic microbial infections.
INTRODUCTION
Neutrophils are the first cells to migrate to the site of infection to
eliminate microorganisms. After activation, neutrophils sequentially discharge granules, which contain a large panel of antimicrobial agents. They also recruit other innate cells including
monocytes and dendritic cells (DCs) at infection sites through
the release of chemokines and of antimicrobial peptides with
chemotactic properties (Scapini et al., 2000).
After pathogen encounter, innate immune cells, including
neutrophils, are activated via a limited number of germline-encoded pattern-recognition receptors (PRRs). Among the bestdocumented PRRs are Toll-like receptors (TLRs) (Akira et al.,
2006). Myeloid differentiation factor-88 (MyD88)-dependent
TLR-triggering leads to cell activation and the release of proinflammatory mediators. Our understanding of non-TLR PRRs
has improved in recent years, and they include RNA helicases,
intracellular sensors for viruses (Kawai and Akira, 2006), and
Nod-like receptors (NLRs) that recognize mostly bacterial
components (Fritz et al., 2006). C-type lectin receptors (CLRs)
make up another large family of cell-surface molecules with
a carbohydrate-recognition domain and contribute mainly to
phagocytosis of microbes, and most of them lack reported
signaling functions. A number of CLR can signal directly or indirectly through an immunoreceptor tyrosine-based activation
motif (ITAM). Clec2 (Suzuki-Inoue et al., 2006) and Clec7A (Dectin-1) (Rogers et al., 2005) can activate cells directly through an
ITAM-like motif in their cytoplasmic region. Alternatively, CLRmediated cell activation may occur indirectly through the interaction of their charged residues in the transmembrane region with
the ITAM-containing DAP12 for Clec5A (MDL-1) (Bakker et al.,
1999) or the FcRg chain for Clec4e (Mincle) (Yamasaki et al.,
2008) and DCAR (Kanazawa et al., 2003). In all cases, it activates
spleen tyrosine kinase (Syk)-dependent signaling pathways in
innate cells. Clec7A (Dectin-1) recognizes b-glucans in fungal
cell walls and is important for antifungal immunity (Saijo et al.,
2007; Taylor et al., 2007). Clec4e (Mincle) also recognizes fungi
Malassezia (Yamasaki et al., 2009), but also Candida albicans,
and contributes to the protection against candidiasis (Wells
et al., 2008). Clec5A (MDL-1) has been recently shown to recognize Dengue virus and to play an important role in the resistance
to infection (Chen et al., 2008).
Most known human TLRs are expressed by neutrophils (Hayashi et al., 2003). Stimulation of human neutrophils with TLR2,
TLR4, TLR5, TLR7, TLR8, and TLR9 agonists induces rapid
activation as evidenced by L-selectin (CD62L) shedding,
CD11b integrin upregulation, IL-8 secretion, and a respiratory
burst (Hayashi et al., 2003). Murine neutrophils express TLR2,
TLR4, and TLR9 mRNAs (Tsuda et al., 2004) and respond to
LPS by shedding L-selectin and upregulation of CD11b (Andonegui et al., 2003). Several studies have identified murine neutrophils as a major source of cytokines, including TNF-a (Bennouna
and Denkers, 2005) and IL-12 (Romani et al., 1997; Bliss et al.,
2000), after stimulation by LPS or Toxoplasma gondii. The mAb
RB6-8C5 (Tepper et al., 1992) has been extensively used for
studying murine neutrophil functions in many murine disease
models. RB6-8C5 binds to Gr-1 (Tepper et al., 1992), corresponding to both Ly6G, a specific granulocyte surface marker,
and Ly6C, an antigen broadly expressed on immune cells,
including neutrophils, eosinophils, monocytes, dendritic cells,
T cells, and NK and NKT cells. However, RB6-8C5 can be
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Bacteria Induce IL-10 Producing Neutrophils
used for in vivo depletion of neutrophils and also to deplete plasmacytoid DCs, T cells, or monocytes, clearly showing that RB68C5 is inappropriate for the study of murine neutrophil functions.
In this work, we used the Ly6G-specific antibodies 1A8
(Fleming et al., 1993) and NIMP-R14 (Lopez et al., 1984) for
a systematic analysis of the innate responses of murine neutrophils to bacteria and to various agonists for PRRs. We demonstrate that highly purified murine neutrophils can be activated
through TLR or CLR pathways, but produced minimal amounts
of proinflammatory cytokines. In contrast, neutrophils produced
large amounts of the anti-inflammatory IL-10 after microbial
stimulation due to coactivation of MyD88 and Syk pathways. In
the context of mycobacterial infections, neutrophils negatively
regulated local lung inflammation in vivo through IL-10 and counteracted the control of the mycobacterial burden during the
chronic phase of the infection. Our findings thus reveal an unexpected and unappreciated role of neutrophils in downmodulating immune responses.
RESULTS
Figure 1. TLR-Activated Neutrophils Are Weak Producers of
Proinflammatory Cytokines
(A) C57BL/6 mouse BM cells were stained with anti-Ly6G, anti-Gr-1,
anti-CD11b, and anti-CD115 and analyzed by flow cytometry. The Gr1hi, Gr1hi
Ly6Ghi, and Gr1hiLy6G populations were further sorted by FACS and stained
with May-Grünwald-Giemsa dye.
(B) Purified BM Ly6Ghi neutrophils were loaded with DHR123 and stimulated
with the indicated TLR agonists for 1 hr to allow detection of respiratory bursts
by flow cytometry.
(C) BM cells were stimulated with various TLR agonists for 6 hr, then intracellular TNF-a and IL-12 were detected by gating on Gr1hi cells.
(D) BM neutrophils (105) and monocytes (5 3 104) were stimulated with various
TLR agonists or 50 ng/ml PMA for 24 hr. TNF-a and IL-12 p40 in culture
supernatants were detected by ELISA. Results are shown as mean of
duplicates ± SD.
762 Immunity 31, 761–771, November 20, 2009 ª2009 Elsevier Inc.
Murine Neutrophils Produce Large Amounts
of IL-10 in the Context of Bacterial Infection
The study of murine neutrophils has been confused by the use of
Gr-1 antibodies (clone RB6-8C5) to identify neutrophils: these
antibodies recognize both Ly6G and Ly6C antigens (Fleming
et al., 1993), and the latter are expressed by monocytes and
dendritic cells. The CD11b+Gr-1hi cells in mouse bone marrow
(BM), blood, spleen, and peritoneum can be clearly separated
into two populations according to Ly6G expression (Figure 1A
and Figure S1 available online): after FACS-sorting, May-Grünwald-Giemsa staining clearly showed that only Gr-1hiLy6Ghi cells
are true neutrophils, whereas most Gr-1hiLy6G cells show
monocyte morphology (Figure 1A and Figure S2). Further analysis showed that both Gr-1hiLy6Ghi and Gr-1hiLy6G cells were
CD11b+, but Gr-1hiLy6Ghi cells were CD115 /low, whereas
Gr-1hiLy6G- cells were strongly CD115+ (Figure 1A and
Figure S1).
We next assessed the capacity of various TLR agonists to activate purified Gr-1hiLy6Ghi neutrophils. Pam3, LPS, and flagellin
readily activated neutrophils in a MyD88-dependent manner,
as shown by their extended morphology, upregulation of
CD11b, downregulation of CD62L, and generation of reactive
oxygen and nitrite intermediates (Figure 1B and Figure S3). We
tested the relative capacity of neutrophils and monocytes to
produce proinflammatory cytokines. Stimulated BM cells were
stained for intracellular TNF-a and IL-12: it was clear that among
Gr1hi cells, monocytes, but not neutrophils, were the major
source of cytokine production in response to agonists for
TLR2, TLR4, TLR5, TLR7, and TLR9 (Figure 1C and Figure S4A).
We then purified neutrophils and monocytes to 99% purity from
BM and found that neutrophils did not produce IL-12 p40
(Figure 1D), IL-1b, IL-6, or CXCL1 (Figure S4B) in response to
any of the TLR agonists; only minute amounts of TNF-a were
detected when neutrophils were stimulated with TLR2 and
TLR4 agonists. In contrast, Gr-1hiLy6G CD115+ monocytes
produced large amounts of TNF-a, IL-12, IL-6, and CXCL1 in
response to all TLR agonists (Figure 1D and Figure S4B). These
results were confirmed with neutrophils purified from blood,
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Bacteria Induce IL-10 Producing Neutrophils
Figure 2. Bacterial Infections Promote an
Anti-inflammatory
Cytokine Response
from Neurophils In Vitro and In Vivo
(A) BM neutrophils and monocytes were stimulated with various TLR agonists. The supernatants
were collected 24 hr later and tested for IL-10.
(B) BM neutrophils were stimulated with live or
heat killed (HK) M. bovis BCG at an MOI of 10,
and then the respiratory burst, CD11b expression
level and IL-10 production were measured.
Shaded, medium alone; red, live BCG; and blue,
heat-killed BCG.
(C) BM neutrophils and monocytes were stimulated with live BCG for 24 hr. Supernatants were
tested for IL-10, TNF-a, IL-1b, IL-6, IL-12 p40,
IL-12 p70, CCL2, CXCL1, and CXCL2.
(D) Neutrophils purified from BM, blood, spleen, or
from peritoneum of thioglycolate-treated (ThioPeri) mice were stimulated with BCG, E. coli
DH5a, S. flexneri, or Pam3 for 24 hr, then assayed
for IL-10. Results are expressed as multiples of the
IL-10 response relative to the response obtained
after Pam3 stimulation.
(E and F) Mice were i.p. infected for 8 hr with BCG
(5 3 107), E. coli DH5a (107) or S. flexneri (107), and
monensin was injected for the last 3 hr. Afterward,
peritoneal Ly6G+ cells were directly analyzed for
intracellular IL-10 (E). Alternatively, peritoneal
neutrophils were purified from mice 6 hr after
infection, and further cultured for 24 hr. Subsequently, supernatants were tested for IL-10 (F).
Results are shown as mean of duplicates ± SD.
spleen, and peritoneum (Figure S5). These various results
show that in the context of TLR activation, murine neutrophils
are efficiently activated, but contribute very much more weakly
than monocytes to proinflammatory cytokine production.
We also found that only Pam3 and LPS stimulated Gr-1hiLy6hi
G neutrophils to produce IL-10, and the production was low
(Figure 2A). Mycobacterium bovis BCG, which contains agonists
for TLR2, TLR4, and TLR9, rapidly activated neutrophils (Figure 2B and data not shown), and neutrophils stimulated with
either live or heat killed (HK) BCG produced large amounts of
IL-10. The production of IL-10 induced by bacteria was at least
10- to 50-fold more than that in response to synthetic TLR
agonists alone (Figure 2 and Figure S6). Monocytes produced
large amounts of proinflammatory mediators TNF-a, IL-1b,
IL-6, IL-12 p40 and p70, CCL2, and CXCL1 but less IL-10
(Figure 2C) in response to BCG. Interestingly, monocytes, but
not neutrophils, from Il10 / mice showed a 10-fold increase in
the production of proinflammatory cytokines in response to
BCG (data not shown), indicating that an autocrine IL-10 regulatory loop controls monocyte response but does not inhibit the
proinflammatory cytokine response of neutrophils. IL-10 production by neutrophils was partially dependent on an IFN-a
and -b autocrine loop (data not shown). When BM neutrophils
were stimulated with Gram-negative bacteria (Escherichia coli
and Shigella flexneri), IL-10 production was greater than that
after stimulation with Pam3 (Figure 2D). Similar results were
obtained with neutrophils isolated from blood and spleen, and
to a lesser extent with thioglycolate-elicited peritoneal neutrophils (Figure 2D). The production of IL-10 by neutrophils was
not influenced by the binding of the Ly6G mAb (Figure S7). We
next assessed the neutrophil response in vivo after the infection
of mice by mycobacteria, Escherichia, or shigella. Using intracellular staining, we found that neutrophils recruited in the peritoneum produced IL-10 after bacterial infection (Figure 2E). We
purified neutrophils from the infected mice and confirmed their
capacity to release IL-10 (Figure 2F). Therefore, after bacterial
stimulation, monocytes express a strong proinflammatory
signature, whereas the neutrophil response is mostly antiinflammatory.
MyD88-TLR2 Is Essential but Not Sufficient
for IL-10 Production by Neutrophils
We next addressed the issue of the signaling pathways required
for the substantial production of IL-10 by murine neutrophils after
bacterial stimulation. TLR agonists are major components of
bacterial cell walls, and accordingly, there was no IL-10 production in neutrophils from Myd88 / mice in response to E. coli,
S. flexneri, and M. bovis BCG (Figure 3). TLR2, but not TLR4
and TLR9, was involved in the neutrophil-derived IL-10 in
response to bacteria. Therefore, IL-10 secretion by bacteriastimulated neutrophils was fully dependent on the TLR2 and
MyD88 pathways, despite the synthetic TLR2 agonist Pam3
being unable to produce the response induced by whole bacteria
(Figures 2D and 2F and Figure S6). IL-10 induction by the TLR2
agonists, lipomannan (LM), and lipoarabinomannan (LAM) from
M. smegmatis either individually or in combination represented
10% of the induction by M. smegmatis (Figure S6). Synergy
between different TLR pathways has been described for proinflammatory responses of DC, but we failed to find any combination of various TLR agonists that enhanced the IL-10 response of
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Bacteria Induce IL-10 Producing Neutrophils
Figure 3. MyD88-TLR2 Signaling Is Essential for IL-10 Production
by Neutrophils after Bacterial Stimulation
BM neutrophils from C57BL/6 (A–C), BALB/c (A), Myd88 / (A and C), Tlr2 /
(B and C), Tlr4 / (B), or Tlr9 / (B) mice were stimulated with BCG (A and B),
E. coli DH5a, S. flexneri, and influenza virus PR8 (C). After 24 hr stimulation,
supernatants were tested for IL-10. Results are shown as mean of duplicates ± SD.
neutrophils (data not shown). Similarly, NOD and TLR pathways
synergize for innate responses (Fritz et al., 2007), but peptidoglycans did not modulate the Pam3-induced IL-10 response of
neutrophils (Figure S8A and data not shown). In conclusion,
TLR2 is necessary but not sufficient for inducing the antiinflammatory response of neutrophils in the context of bacterial
stimulation.
C-Type Lectin Agonists Synergize with TLR2 to Promote
the Anti-inflammatory Signature of Neutrophils
Another important large family of PRRs, C-type lectins, plays an
important role in innate defenses. We first screened for the
expression of various lectins and scavenger receptors by neutrophils (Figure 4A). CD204, CD205, Clec7A (Dectin-1), and Clec5A
(MDL-1) are expressed by neutrophils. We coated antibodies
specific for all these lectins and assessed the capacity of crosslinked CLR to activate neutrophils. Under these conditions, only
anti-Clec5A activated neutrophils as judged by phenotypical
changes (data not shown), but they failed to induce IL-10
(Figure 4B and Figure S8B). However, Clec5A antibodies, either
monoclonal or polyclonal, combined with Pam3 induced strong
IL-10 production, as bacteria do. Given that Clec7A antibodies
block ligand binding without cell signaling, we stimulated neutrophils with curdlan (a high MW b1-3glucan from agrobacteria),
a natural agonist of Clec7A. Like TLR agonists, curdlan strongly
activated neutrophils to upregulate CD11b, to shed CD62L, and
to generate a respiratory burst (Figure S9A), but induced only
minimal production of cytokines and chemokines, including
IL-10, TNF-a, and CXCL2 (Figure 4C and Figures S8A and
S9B). Curdlan-activated monocytes produced TNF-a, IL-6,
IL-12, CCL2, CXCL1, and CXCL2, and the production was further
increased by the addition of Pam3 (Figure S9B). Pam3 plus
764 Immunity 31, 761–771, November 20, 2009 ª2009 Elsevier Inc.
curdlan did not modify the cytokine and chemokine profile of
the neutrophil response, but—similar to bacteria—potently drove
neutrophils to produce substantial amounts of IL-10 and CXCL2
(Figure 4C and Figures S8A and S9B). LPS, but not TLR7 or TLR9
agonists, also synergized with curdlan to induce IL-10 production
by neutrophils (data not shown). The NOD2 agonist MDP
combined with curdlan had no effect on IL-10 production by
neutrophils (Figure S8A). We also tested A. fumigatus as another
source of b-glucans to stimulate neutrophils. Again, fungi alone
induced minute amounts of IL-10, and induction was greatly
enhanced by the presence of a TLR2 agonist (Figure S8C). Very
recently, the mycobacterial factor trehalose-6,6 dimycolate
(TDM) was shown to stimulate the innate responses of DCs and
macrophages in a FcRg chain-dependent manner (Werninghaus
et al., 2009). When we stimulated neutrophils with Pam3 together
with TDM from M. tuberculosis, they readily produced large
amounts of IL-10 (Figure 4D). The influence of the FcRg pathway
was further confirmed with immune complexes that substantially
enhanced IL-10 production in Pam3-stimulated neutrophils
(Figure S8D). Altogether, these results demonstrate that activation through selective TLR together with Clec5A and Clec7A pathways or with carbohydrate compounds can switch neutrophils to
become high IL-10 producers.
FcRg, DAP12, and Syk Signaling Contribute
to Bacterial Induction of IL-10 by Neutrophils
To discriminate between the different CLR signaling pathways
that may contribute to IL-10 production after bacterial stimulation, we tested the response of neutrophils from FcRg-deficient
(Fcer1g / ) mice (Figure 4E) and from DAP12KDY75 mice
(Figure 4F), a loss-of-function mutant of DAP12 (Tomasello
et al., 2000). We first stimulated WT and Fcer1g / neutrophils
with Pam3 alone or in combination with glycans. Fcer1g /
neutrophils failed to respond to mycobacterial TDM glycolipid;
however, they responded well to b-glucans or DAP12-dependent
Clec5A stimulation. Under the same conditions, DAP12KDY75
neutrophils failed to respond to Clec5A triggering, whereas their
production of IL-10 was strongly stimulated in response to curdlan and TDM. After bacterial stimulation, DAP12 was mainly,
but not solely, involved in the response to S. flexneri and E. coli,
whereas the lack of FcRg did not affect the response. The
response to BCG was mainly dependent on DAP12, and FcRg
made a minor contribution, indicating that TDM is not the major
active mycobacterial component. Clec7A (Dectin-1) has been
implicated in the interaction between mycobacteria and both
dendritic cells and macrophages (Yadav and Schorey, 2006);
however, IL-10 production in response to M. bovis BCG was
only slightly inhibited by laminarin, a low MW b-glucan with
Clec7A-blocking properties (Gantner et al., 2003) (Figure 4G). In
contrast, laminarin inhibited activation due to S. flexneri by
50%. FcRg and DAP12 signaling, as well as Clec5A- (Bakker
et al., 1999) and Clec7A-mediated activation (Rogers et al.,
2005), involve Syk tyrosine kinase, which is essential for downstream inflammatory cytokine responses. We found that the
Syk tyrosine kinase inhibitor piceatannol and Syk inhibitors III
halved IL-10 production by neutrophils after stimulation by
S. flexneri, E. coli, or Pam3 combined with curdlan or anti-Clec5A
(Figure 4G). It is unclear whether the partial inhibition observed
was due to the limited efficacy of these pharmacological
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Bacteria Induce IL-10 Producing Neutrophils
Figure 4. C-Type Lectins Synergize with
TLR2 but Differentially Require FcRg,
DAP12, or Syk Pathways to Promote the
Anti-inflammatory Activity of Neutrophils
(A) Analysis of the expression profile for C-type
lectins and scavenger receptors on BM neutrophils by FACS (thin line).
(B–F) BM neutrophils were stimulated with immobilized anti-Clec5A mAb (B), curdlan (C), and
TDM from M. tuberculosis (D) in the presence or
absence of Pam3. BM neutrophils from WT
(E–G), Fcer1g / (E), and DAP12KDY75 (F) were
stimulated as indicated, and the supernatants
were tested for IL-10. Results are shown as
mean of duplicates ± SD.
(G) BM neutrophils were pretreated with 500 mg/ml
laminarin or Syk inhibitors at 37 C for 1 hr, then
stimulated as indicated. Results for the IL-10
responses in the presence of inhibitors are expressed as percentages of that in the absence of
inhibitors. Results are shown as mean of three
independent experiments ± SD. Not done, N.D.
inhibitors and would need further investigation with Syk-deficient
neutrophils. These inhibitors fully inhibited activation by M. bovis
BCG, but no inhibition was observed after Pam3 stimulation. This
series of experiments provides strong evidence that the antiinflammatory properties of neutrophils are engaged by synergy
between the MyD88-TLR and Syk pathways.
TLR2 and CLR-Syk Sustain Phosphorylation
of the p38 and Akt for IL-10 Production by Neutrophils
The regulation of IL-10 transcription is a complex process, and
most relevant work has been with macrophages or GMCSFderived DC. These studies found that the activation of MAP
kinase pathways, particularly p38 and ERK kinases, are essential
for IL-10 production by innate cells (Lucas et al., 2005; Yi et al.,
2002). We therefore used inhibitors specific for p38, ERK, and
JNK kinases to test whether MAP kinases are also important
for IL-10 production by neutrophils. We found that only the p38
kinase inhibitor SB203580 strongly inhibited the IL-10 production by neutrophils in various experimental conditions
(Figure 5A). In contrast, SB203580 had
a less obvious effect on TNF-a production
(data not shown). The phosphatidylinositol-3 kinase (PI3K)-Akt pathway can also
contribute to IL-10 production (Pengal
et al., 2006): we found that LY294002,
an inhibitor of PI3K, inhibited neutrophil
production of IL-10 in response to
bacteria as well as to single TLR and
CLR agonists (Figure 5B). The doses of
pharmacological inhibitors used did not
affect neutrophil viability (Figure S10).
To explore further the kinase pathways,
we monitored the phosphorylation of
p38 and Akt kinases during neutrophil
activation. After 30 min, p38 and Akt
phosphorylation was strongly induced
by Clec5A and Clec7A agonists, whereas only a modest phosphorylation was observed after TLR2 activation (Figures 5C
and 5D). Interestingly, kinetic studies revealed that CLR-induced
p38 and Akt activation quickly receded in neutrophils, but was
maintained in the presence of Pam3 stimulation (Figure 5D). It
remains to be determined whether Pam3 stimulation inhibits
dephosphorylation of the proteins phosphorylated by CLR
signaling or induces new phosphorylation. These observations
show that activation of p38 MAP and PI3-Akt kinases is essential
for IL-10 production by neutrophils after TLR and/or CLR stimulation and that sustained kinase activation may contribute to the
high amount of IL-10 secretion.
Neutrophils Control Local Lung Inflammation
In Vivo after Mycobacterial Infection
To investigate directly the role of neutrophils in the regulation of
innate responses in vivo, we studied the influence of neutrophils
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Bacteria Induce IL-10 Producing Neutrophils
Figure 5. The p38 MAPK and Akt Kinase
Pathways Are Essential for IL-10 Production
by Neutrophils
(A and B) BM neutrophils were pretreated with
inhibitors for p38 (SB203580), ERK (U0126), JNK
(SP600125), and PI3 kinase (LY294002) or with
DMSO at 37 C for 1 hr and then stimulated as indicated. Twenty-four hours later, supernatants were
tested for IL-10. The results for the IL-10
responses in the presence of inhibitors are expressed as percentages of that in the absence of
inhibitors. Results are shown as mean of three
independent experiments ± SD.
(C and D) BM neutrophils were stimulated as indicated for 30 min (C) or for different time periods
(D); then, intracellular phosphorylated p38 MAPK
and Akt kinases (bold lines) were detected by
flow cytometry. Shaded histograms correspond
to Ig isotype controls.
in the development of lung inflammation induced by BCG. Intranasally, BCG infection in mice led to a rapid recruitment of
neutrophils into the lung alveolar cavity (as early as 6 hr postinfection; Figure 6A). These in vivo-activated neutrophils secreted
IL-10 after 24 hr ex vivo culture. Neutrophils from the alveolar
cavity produced more IL-10 than did neutrophils from lung
parenchyma (Figure 6B).
To test the influence of neutrophils on the inflammatory
response in the lung, we first depleted neutrophils by using
Ly6G mAb and then administered BCG intranasally: neutrophil
infiltration into the lungs was strongly reduced, without affecting
the number of monocytes, macrophages, and DCs (Figure S11).
Neutrophil depletion led to a reduced amount of IL-10 and
increased TNF-a produced by lung cells, without affecting the
mycobacterial load (Figure 6C). One possible explanation would
be that the loss of direct killing activity of neutrophil could
766 Immunity 31, 761–771, November 20, 2009 ª2009 Elsevier Inc.
compensate the increase of inflammation
resulting in a neutral effect on the control
of the acute phase of infection. To test
this hypothesis, we depleted neutrophils
in Il10 / mice prior BCG infection. In
these conditions, lack of neutrophils led
to an increase of mycobacterial load, indicating the sole loss of neutrophil bacterial
killing activity (Figure 6D). These results
underline the dual role of neutrophils in
acute infection, a direct antimicrobial
activity counterbalanced by anti-inflammatory properties. To confirm the latter
point and to clarify the impact of neutrophil-derived IL-10, we transferred WT or
Il10 / neutrophils into Il10 / recipient
mice and then challenged these mice
with BCG. Transfer of Il10 / , but not
WT, neutrophils decreased the mycobacterial load as compared to the controls
(Figure 6E). These data indicate that in
the absence of anti-inflammatory properties, transferred neutrophils contribute
directly to mycobacterial elimination. In contrast, when WT
neutrophils are transferred, their dual influence on direct bacterial elimination and on inflammation leads to a neutral effect for
mycobateria control, in accordance with the neutral effect
observed in neutrophil-depleted WT mice (Figure 6C). Importantly, WT and Il10 / transferred neutrophils were similarly
recruited in the lungs after infection (Figures 6F and 6G). IL-10
was only detected in mice receiving WT neutrophils (Figure 6H). The production of TNF-a by macrophages, monocytes,
and DCs was much lower than that untreated control mice
in Il10 / mice that had received wild-type, but not Il10 / ,
neutrophils (Figure 6I). In an identical experimental system,
BAL was assayed for proinflammatory cytokines. The BCG-infected mice given WT neutrophils produced much less TNF-a,
IL-1b, and IL-6 than those given Il10 / neutrophils (Figure 6J).
These results clearly show that neutrophil-derived IL-10 can
Immunity
Bacteria Induce IL-10 Producing Neutrophils
Figure 6. Neutrophils Control Local Lung Inflammation in an IL-10-Dependent Manner after Intranasal Inoculation with M. bovis BCG
(A) Kinetics of neutrophil infiltration into the alveolar cavity after intranasal (i.n.) inoculation of B6 mice (n = 3–6) with 8 3 106 CFU of BCG. Results are shown as
mean of each group of mice ± SD.
(B) Neutrophils were purified from BAL or lung parenchyma, 6 hr after intranasal administration of BCG, cultured for 24 hr, and tested for IL-10 production.
Total-lung neutrophils from PBS-treated mice were used as controls. Results are shown as mean of duplicates ± SD.
(C) B6 mice were PMN depleted with Ly6G mAb 1 day before i.n. inoculation with BCG. Whole-lung cells were collected and further cultured for 24 hr and tested
for IL-10 and TNF-a production; BCG CFU lungs were counted at day 3 postinfection.
(D) Il10 / mice were PMN depleted or not before BCG challenge, and CFUs were counted in the lungs.
(E–J) A total of 5 3 106 B6 or Il10 / neutrophils or PBS were i.n. transferred into Il10 / mice before BCG challenge. As shown in (E), CFUs were counted in the
lungs. (F) shows tissue sections of lungs (nuclei in blue) after transfer of PE-labeled neutrophils (in red) and after BCG challenge or with no treatment (inset). (G)
shows analysis of neutrophil migration to the lungs after transfer of a 1:1 mixture of B6 and Il10 / PE-labeled neutrophils. (H) shows IL-10 detection in lungs from
mice administered WT or Il10 / neutrophils. Results are shown as mean of duplicates ± SD. As shown in (I), lung macrophages (MF), monocytes (Mo), and DCs
were tested for intracellular TNF-a. As shown in (J), BALs were analyzed for TNF-a, IL-1b, and IL-6. Six mice were used for each group. Results are shown as mean
of each group of mice ± SD. *p < 0.05 compared to other groups.
downmodulate local lung inflammation induced by mycobacteria.
Neutrophils Influence Chronic Infection
by Virulent Mycobacteria
Finally, we evaluated the role of neutrophils in the context of
a chronic infection by virulent M. tuberculosis H37Rv. In vitro,
neutrophils infected with H37Rv produced, in a MyD88-dependent manner (data not shown), large amounts of IL-10 (Figure 7A).
We then infected mice with an aerosol of M. tuberculosis H37Rv
(Mtb, 100 CFU per mouse). Five weeks later, we purified and
cultured neutrophils without additional stimulation. Under these
conditions, lung neutrophils isolated from both BAL and parenchyma of Mtb-infected mice produced IL-10 (Figure 7B). To
Immunity 31, 761–771, November 20, 2009 ª2009 Elsevier Inc. 767
Immunity
Bacteria Induce IL-10 Producing Neutrophils
Figure 7. Neutrophils Lower Immunity
during M. tuberculosis Chronic Infection
(A) BM neutrophils from BALB/c mice were stimulated with live M. tuberculosis H37Rv for 24 hr, and
supernatants were tested for IL-10.
(B) BALB/c mice were aerosolly infected with a
M. tuberculosis H37Rv. Five weeks later, 2 3 105
neutrophils purified from lungs and BM were
cultured ex vivo for 24 hr. Alternatively, Ly6G+ cells
were purified independently from BAL and lung
parenchyma of infected mice (n = 4). The supernatants were tested for IL-10.
(C) BALB/c mice were infected with M. tuberculosis H37Rv by the aerosol route. Five weeks later,
mice received two intravenous injections of antiLy6G mAb (NIMP-R14) or Ig control 3 days apart.
(C and D) One day after each injection, lungs cells
were analyzed phenotypically and for IL-10 and
TNF-a release (n = 3 mice/group).
(E) Alternatively, whole-lung homogenates after
two injections were tested for IL-6, IL-17, and
IFN-g (n = 6 mice/group).
(F) Infected mice (n = 10/group) received three
injections of depleting and control antibody, then
H37Rv CFU were counted in the lungs. Results
are representative of at least two experiments.
*p < 0.05.
identify the effects of neutrophils on inflammation and Mtb infection, we depleted neutrophils from chronically infected mice by
injection of Ly6G mAb. After this treatment, the number of monocytes slightly decreased, whereas inflammatory DCs increased
(Figure 7C). When we cultured cells from infected lungs
ex vivo, we found a significant (p < 0.05) reduction of IL-10
release and slight increase of TNF-a in depleted mice as
compared to control infected mice (Figure 7D). In addition,
concentrations of IL-6 and IL-17, but not IFN-g, were higher in
whole-lung homogenates from neutrophil-depleted infected
mice than those from infected control isotype mice (Figure 7E).
Concommitantly, 1 week after neutrophil depletion, the number
of mycobacterial CFU in the lung was 2.5-fold lower than in
controls (Figure 7F). These experiments indicate that virulent
mycobacteria can activate neutrophils to produce IL-10 and
that neutrophils contribute to the persistence of a high mycobacterial burden during chronic infection by M. tuberculosis H37Rv.
DISCUSSION
In this study, we systemically studied the innate responses of
murine neutrophils in response to single agonists for TLR,
NLR, and CLR, as well as in response to whole bacteria (Mycobacteria, E. coli, and S. flexneri), fungus (A. fumigatus), and
virus (influenza virus). We report several interesting findings:
First, murine neutrophils give no or weak proinflammatory
responses to stimulation with single agonists or microorgan768 Immunity 31, 761–771, November 20, 2009 ª2009 Elsevier Inc.
isms; second, stimulation with bacteria
and combined TLR/CLR-Syk activation
trigger a predominantly anti-inflammatory response in neutrophils involving
substantial IL-10 production; and third,
neutrophils negatively regulate lung inflammation induced by
mycobacterial infection in vivo.
Murine neutrophils were readily activated in a MyD88-dependent manner by TLR2, TLR4, and TLR5, but not by TLR3, TLR7,
or TLR9 agonists, but only produced minute amounts of proinflammatory cytokines; this finding is not consistent with previous
reports (Bennouna and Denkers, 2005; Bliss et al., 2000). AntiGr-1 (Ly6G and C) has been routinely used for identifying murine
neutrophils in numerous studies, but Gr-1 is also expressed on
many other cells; particularly relevant is a population of Gr-1hi
inflammatory monocytes (Geissmann et al., 2003), which, as
we demonstrated here, have a substantial capacity to produce
proinflammatory cytokines. Therefore, our work suggests that
many of the functions of murine neutrophils described in early
studies should be interpreted with caution and need to be reconsidered. We show here that neutrophils preferentially produce
IL-10, rather than inflammatory cytokines, in response to
bacteria and do so in a MyD88- and Syk-dependent manner.
Coligation of multiple PRRs corresponds to the physiologically
relevant innate cell activation by microorganisms, and synergistic effects have been observed between different TLR
agonists and between TLR and other PRRs. The TLR7 or TLR8
agonist synergizes with TLR3 or TLR4 agonists to induce strong
production of IL-12 p70 by DCs (Napolitani et al., 2005). The TLR
and NLR pathways also synergize for inflammatory responses
(Fritz et al., 2006). However, we did not observe any such
synergy for IL-10 production by neutrophils. The relationship
Immunity
Bacteria Induce IL-10 Producing Neutrophils
between TLR and ITAM-activating receptors is less clear, with
respect to either synergistic or inhibitory outcomes. Macrophages deficient for DAP12 show an enhanced response to
TLRs probably because of basal ITAM-Syk activation in these
cells (Hamerman et al., 2005). Ligation of DAP12-associated
receptors in myeloid cells leads to the production of proinflammatory cytokines (Bouchon et al., 2001; Turnbull et al., 2005),
but may also dampen the TLR response of macrophages
(Hamerman et al., 2006). Fcer1g / and DAP12KDY75 neutrophils
showed a slightly greater IL-10 response to TLR2 activation than
WT neutrophils, but a lower response to bacteria. Therefore, the
net effect of TLR2 and CLR-Syk coactivation or bacterial stimulation was a strong enhancement of the IL-10 response. The
collaboration of TLR and Clec7A (Dectin-1) pathways has been
shown to induce an increased inflammatory response in both
macrophages and DCs (Gantner et al., 2003), although Clec7A
(Dectin-1) together with CD40 also potently induces IL-10 from
DCs (Rogers et al., 2005). In our study, bacteria induced strong
IL-10 production by neutrophils, and this phenomenon was
dependent on TLR2 and Syk coactivation. Clearly, different
PAMPs and PRRs are involved in the Syk activation process
because the role of DAP12 and FcRg was dependent on the
bacteria tested. Full inhibition of mycobacteria-induced neutrophil-derived IL-10 was achieved with Syk inhibitors, but only
50% inhibition was obtained in the context of shigella stimulation. In addition, half of the IL-10 response was dependent on
DAP12 for all bacteria tested, whereas a role for FcRg was
only evidenced for mycobacteria. Coengagement of TLR2 and
Clec5A also promoted regulatory neutrophils, and recognition
of Dengue virus by Clec5A mainly involves fucose residues
(Chen et al., 2008). This phenomenon was also successfully
mimicked by a combination of TLR2 and b-glucans. All these
stimuli preferentially induced substantial IL-10 production by
neutrophils, with only small or undetectable amounts of proinflammatory cytokines. This is in sharp contrast with monocytes
that display a strong proinflammatory profile in the same conditions. Importantly, the proinflammatory response of monocytes
is under the control of an autocrine IL-10 regulatory loop,
whereas neutrophils are not sensitive to IL-10, and thus have
a true anti-inflammatory phenotype. This indicates that, in addition to their microbicidal activities, neutrophils can turn into
a regulatory cell type upon encountering a pathogen. DAP12deficient mice are more susceptible to endotoxin shock and
more resistant to Listeria infection (Hamerman et al., 2006). In
view of the innate anti-inflammatory response of neutrophils,
the lack of DAP12 signaling may also play a role in inflammation
and resistance to bacterial infection in vivo.
Neutrophils have been described as supporters of proinflammatory responses (Ricevuti, 1997) and as able to exert their
effects directly on DCs (Bennouna et al., 2003; van Gisbergen
et al., 2005). However, activated neutrophils inhibit T cell functions and suppress cytokine production through the generation
of hydrogen peroxide (Schmielau and Finn, 2001). Neutrophils
purified from mice suffering of systemic inflammatory response
syndrome display regulatory features that can convert resident
macrophages into alternatively activated macrophages that
usually contribute to chronic infections (Tsuda et al., 2004). Our
work unequivocally demonstrates a central regulatory role of
neutrophils in dampening the proinflammatory response.
In the context of bacterial infection, this phenomenon counterbalances the high proinflammatory activity of other innate cells
such as monocytes, macrophages, and DCs. We confirmed
this finding by showing that neutrophils control local lung innate
inflammation after mucosal M. bovis BCG infection. In the acute
phase of the infection, the global activity of neutrophils is
a balance between the positive (direct bacterial elimination)
versus the negative (anti-inflammatory) properties of neutrophils
without much influencing the mycobacterial growth. However, in
the chronic phase of M. tuberculosis infection, i.e., with a high
and stable mycobacterial load, we found that neutrophil depletion was beneficial to the host and was associated with
increased amounts of IL-6 and IL-17, but not IFN-g in the
infected lungs. IL-6 is upstream from Th17 cell differentiation
(Dong, 2008) and both Th1 and Th17 cells are associated with
M. tuberculosis control (Khader et al., 2007); this type of
phenomenon may also be relevant for other bacterial infections.
Interestingly, one of the rare descriptions of IL-10 production
by human neutrophils concerns those infiltrating UV-irradiated
skin (Piskin et al., 2005). Because we found that TLR2 and CLR
coactivation leads to IL-10 production by neutrophils, it is
possible that endogenous ligands for these receptors may be
involved in this phenomenon. In addition to C. albicans recognition (Wells et al., 2008), Clec4e (Mincle), a DAP12-dependent
CLR, recognizes SP130 nuclear protein from damaged cells
(Yamasaki et al., 2008). Likewise, extracellular matrix components, such as hyaluronan (Scheibner et al., 2006) and versican
(Kim et al., 2009), can activate innate inflammation through
TLR2. Therefore, endogenous TLR2 ligands may act in concert
with CLR ligands released by damaged cells to activate antiinflammatory neutrophils. Similar phenomena may contribute
to the activity of Gr1-positive myeloid-derived suppressor cells
that are involved in tumor immune suppression (Bronte et al.,
2003).
In summary, we have identified a unique regulatory role for
neutrophils triggered by mycobacteria and Gram-negative
bacteria that is based on co-activation through the MyD88 and
CLR-DAP12-Syk pathways; this regulation involves the production of large amounts of IL-10. IL-10-producing neutrophils are
able to temper lung inflammation upon mycobacterial infection
in vivo and may play a similar role in other bacterial infections.
EXPERIMENTAL PROCEDURES
Mice
C57BL/6 and BALB/c adult mice were purchased from Janvier, and B10D2
from Jackson Laboratories. Mice deficient for IL-10, TLR2, TLR4, TLR9, type
I IFN receptor, and FcRg were all on a C57BL/6 background and bred in our
animal facilities. DAP12 DY75 mice, loss-of-function mutant of DAP12, were
on a B10D2 background (Tomasello et al., 2000). The mice were housed
on-site in specific pathogen-free conditions and used when they were
6–10 weeks old. Animal studies were approved by the Institut Pasteur safety
committee in accordance with French and European guidelines.
Cell Purification and FACS Analysis
Neutrophils and monocytes were purified from BM, lungs, spleen, blood, or
thioglycolate-treated peritoneum. For neutrophils, cells from organs were
stained with biotin- or PE-anti Ly6G, stained with anti-biotin or anti-PE beads
(Miltenyi Biotec), and positively selected on an automated magnetic cell sorter
(AutoMACSpro; Miltenyi Biotec). In some experiments, BM cells were directly
stained with FITC-anti Ly6G, PE-anti CD115, and APC-anti CD11b, then
Immunity 31, 761–771, November 20, 2009 ª2009 Elsevier Inc. 769
Immunity
Bacteria Induce IL-10 Producing Neutrophils
neutrophils (Ly6GhiCD11b+CD115 ) and monocytes (CD115+CD11b+Ly6G )
were sorted by flow cytometry on a FACSAria apparatus (BD Biosciences).
FACS analysis was performed with Flowjo software (Tree Star).
Neutrophil Responses
A total of 105 or 2 3 105 neutrophils and/or the indicated number of monocytes
were cultured in RPMI-1640 complete medium in 96-well flat-bottom plates
and stimulated with various stimuli for 24 hr. The supernatants were collected
and assayed for cytokines. IL-10, TNF-a, IL-1b, IL-6, IL-12 p40, and IL-12 p70
were determined by standard sandwich ELISA with appropriate Ab pairs (all
from BD Biosciences). Chemokines CCL2, CXCL1, and CXCL2 were detected
with a multiplex kit (Invitrogen). For intracellular cytokine staining for TNF-a,
IL-12, and IL-10, cells were stimulated as indicated in figure legends, brefeldin
A was added for the last 3 hr during stimulation, and the cells were processed
in accordance with the manufacturer’s protocol (BD Biosciences). For detecting reactive oxygen intermediates, purified neutrophils were loaded with 5 mM
Dihydrorhodamine 123 (Invitrogen) for 15 min at 37 C and then stimulated with
various stimuli for the indicated time. For monitoring intracellular phosphorylated kinases, purified neutrophils were stimulated as indicated, then fixed
with BD Phosflow Lyse/Fix Buffer I at 37 C for 10 min, permeabilized with
BD Phosflow Perm Buffer III for 30 min on ice, and stained.
For inhibition experiments, purified BM neutrophils were pretreated with
500 mg/mL laminarin (Sigma) or 1 mM of the various kinase inhibitors at 37 C
for 1 hr. Afterward, the cells were stimulated with various agents for 24 hr
and the supernatants were tested for IL-10. The kinase inhibitors used were
the following: SB203580 (p38), U0126 (ERK), SP600125 (JNK), LY294002
(PI3K), piceatannol, and Syk inhibitor III (Syk), all from Calbiochem Merck.
Mycobacterial Infections
The mice were inoculated intranasally with 8 3 106 CFU M. bovis BCG and the
first BAL wash in 1 ml PBS was tested for cytokines. The lung parenchyma was
digested with collagenase and DNase I at 37 C for 30 min so that single-cell
suspensions could be obtained. Cells from BAL fluids and lung parenchyma
were further incubated for 3 hr in the presence of brefeldin A for intracellular
TNF-a detection. Alternatively, neutrophils were purified from lung cell suspensions for tests for IL-10 production. In brief, cell suspensions were loaded onto
Percoll gradient media (80%–65%–55%) and after centrifugation, cells at the
80%–65% and 65%–55% interfaces were collected. Lung neutrophils were
purified as described above with PE anti-Ly6G and anti-PE beads. Neutrophil
depletion was achieved by intravenous (i.v.) injection of 200 mg anti-Ly6G
(either clone 1A8 or NIMP-R14, with similar results) mAb or isotype control
antibody 1 day before BCG administration. Purified 1A8 mAb was obtained
from BioXcell, and NIMP-R14 mAb was prepared from the eponymous cell
clone kindly given by G. Millon (Institut Pasteur).
For M. tuberculosis H37Rv infection, BALB/c mice were infected via the aerosol route with a customized apparatus for delivering a retained inhaled dose
of 100 ± 10 CFU. For neutrophil depletion, mice received two to three injections
of 200 mg anti-Ly6G (NIMP-R14) mAb or isotype control antibody as indicated in
the legends. Five to six weeks after infection, lungs were collected and either
cells were recovered for FACS analysis and culture or whole-lung homogenates were prepared for cytokine detection and H37Rv CFU counting. Alternatively, lung neutrophils were purified and cultured for 24 hr.
Statistical Analysis
Unpaired t test was used for comparisons between two groups (data from
such experiments are presented as mean values ± SD). p values of < 0.05
were considered statistically significant.
SUPPLEMENTAL DATA
Supplemental Data include Supplemental Experimental Procedures and
11 Figures and can be found with this article online at http://www.cell.com/
immunity/supplemental/S1074-7613(09)00456-7.
ACKNOWLEDGMENTS
We are grateful to E. Vivier and M. Dalod for DAP12 KDY75 mice, and to
P. Bruhns for Fcer1g / mice. We thank O. Granet for A. fumigatus, G. Sellge
770 Immunity 31, 761–771, November 20, 2009 ª2009 Elsevier Inc.
for S. flexneri, R. Brosch for M. smegmatis, and C. Fayolle for NIMP-R14 mAb.
X. Z. was supported by the European Commission (Cellprom-NMP4-CT-2004500039) and by Institut Pasteur (PTR260). This work was supported by a grant
from Agence Nationale de la Recherche (MIME 2006) and by the Ligue du
Cancer.
Received: April 3, 2009
Revised: June 20, 2009
Accepted: September 9, 2009
Published online: November 12, 2009
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