This information is current as
of June 16, 2017.
IL-17A Produced by Neutrophils Protects
against Pneumonic Plague through
Orchestrating IFN-γ−Activated Macrophage
Programming
Yujing Bi, Jiyuan Zhou, Hui Yang, Xiao Wang, Xuecan
Zhang, Qiong Wang, Xiaohong Wu, Yanping Han, Yajun
Song, Yafang Tan, Zongmin Du, Huiying Yang, Dongsheng
Zhou, Yujun Cui, Lei Zhou, Yanfeng Yan, Pingping Zhang,
Zhaobiao Guo, Xiaoyi Wang, Guangwei Liu and Ruifu Yang
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The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2014 by The American Association of
Immunologists, Inc. All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2014; 192:704-713; Prepublished online 13
December 2013;
doi: 10.4049/jimmunol.1301687
http://www.jimmunol.org/content/192/2/704
The Journal of Immunology
IL-17A Produced by Neutrophils Protects against Pneumonic
Plague through Orchestrating IFN-g–Activated Macrophage
Programming
Yujing Bi,*,1 Jiyuan Zhou,*,1 Hui Yang,†,‡,1 Xiao Wang,†,‡,1 Xuecan Zhang,* Qiong Wang,*
Xiaohong Wu,* Yanping Han,* Yajun Song,* Yafang Tan,* Zongmin Du,* Huiying Yang,*
Dongsheng Zhou,* Yujun Cui,* Lei Zhou,* Yanfeng Yan,* Pingping Zhang,* Zhaobiao Guo,*
Xiaoyi Wang,* Guangwei Liu,†,‡ and Ruifu Yang*
Y
ersinia is a genus that comprises three human pathogen
species. Y. enterocolitica and Y. pseudotuberculosis are
enteropathogens that cause gastrointestinal diseases, and
Y. pestis is the causative agent of plague (1, 2). In general, plague is
*State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing 100071, China; †Key Laboratory of Medical Molecular Virology of Ministry Of Education/Ministry of Health, Department of Immunology,
School of Basic Medical Sciences, Fudan University, Shanghai 200032, China; and
‡
Biotherapy Research Center, Fudan University, Shanghai 200032, China
1
Y.B., J.Z., H.Y., and X.W. contributed equally to this study as cofirst authors.
Received for publication June 26, 2013. Accepted for publication November 8, 2013.
This work was supported by National Natural Science Foundation for General Programs of China Grants 81271907 (to Y.B.) and 31171407 and 81273201 (to G.L.) and
by Key Basic Research Project of the Science and Technology Commission of
Shanghai Municipality Grant 12JC1400900 (to G.L.).
Y.B. designed and did the experiments with cells and mice, analyzed data, and
contributed to writing the manuscript; J.Z. designed the experiments with mice and
contributed to discussion; H.Y. and X.W. analyzed histology data; X.Z., Q.W., X.W.,
and Y.H. did the experiments with mice and contributed to managing the mouse
colonies; Y.S., Y.T., Z.D., Y.C., L.Z., Y.Y., P.Z., and Z.G. contributed to discussion;
X.W. analyzed data and contributed to writing the manuscript; G.L. designed experiments, analyzed data, and wrote the manuscript; and R.Y. designed experiments,
analyzed data, wrote the manuscript, and provided overall direction.
Address correspondence and reprint requests to Dr. Ruifu Yang, Dr. Guangwei Liu, or
Xiaoyi Wang, State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of
Microbiology and Epidemiology, Beijing 100071, China (R.Y. and X.W.), or Key
Laboratory of Medical Molecular Virology of Ministry of Education/Ministry of
Health, Department of Immunology, School of Basic Medical Sciences, Fudan University, Shanghai 200032, China (G.L.). E-mail addresses: [email protected]
(R.Y.), [email protected] (G.L.), and [email protected] (X.W.)
The online version of this article contains supplemental material.
Abbreviations used in this article: BALF, bronchoalveolar lavage fluid; CL, clodronate; iNOS, inducible NO synthase; KO, knockout; Lipo, liposome; rm, recombinant
murine; WT, wild-type.
Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1301687
manifested as either a bubonic or a pneumonic form. Bubonic plague
occurs when Y. pestis is inoculated in the host by the bite of an
infected flea, and this process is considered one of the most common presentations of the disease (3). When inhaled, Y. pestis causes
primary pneumonic plague, a contagious disease that can be spread
via respiratory droplets (1). The primary pneumonic plague is a
rapidly progressive and aggressive pneumonia that usually develops
in 2–3 d after exposure, with mortality rates approaching 100% if
effective treatment is delayed (4, 5). However, little is known about
innate host responses in the pathogenesis of the pneumonic plague
at an early stage.
IL-17 is an important cytokine mainly produced by Th17, which
has a major function in autoimmunity that results from a dysfunction
of adaptive immunity (6–8). Although most data have suggested that
the Th17/IL-17 pathway’s functions in mediating chronic inflammatory diseases, such as rheumatoid arthritis (8, 9), asthma (10, 11),
systemic lupus erythematosus (6), multiple sclerosis (12), and allograft rejection (13), other findings have indicated that such a
pathway may participate in innate immunity (14). Acute bacterial
inflammation usually recruits neutrophils with a subsequent activation
of macrophages, thereby providing protection against foreign bacterial
infection. However, the mechanism involved in this activation remains
unclear.
In pneumonic plague, a model of acute bacterial infection, IL-17
pathways appear to be activated (1). Infected lung injury causes
production of IL-17–promoting cytokines, such as TGF-b, IL-6,
TNF-a, and IL-1b, in the inflamed lung (1). In addition, Th1,
Th17, and neutrophils are important in the pathogenesis of autoimmune inflammation (15–17). Recently, some studies have examined
the function of IL-17 in innate immunity, including in infected lungs
(18, 19). Our study demonstrated that IL-17 pathways are activated
in pneumonic plague. We also showed that IL-17A produced by
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Innate immune cells, including neutrophils and macrophages, are critically involved in host antimicrobial defense responses. Intrinsic
regulatory mechanisms controlling neutrophil and macrophage activities are poorly defined. In this study, we found that IL-17A, a natural
signal factor, could provide protection against early pneumonic plague inflammation by coordinating the functions of neutrophils and
programming of macrophages. The IL-17A level is promptly increased during the initial infection. Importantly, abrogation of IL-17A or
IL-17AR significantly aggravated the infection, but mIL-17A treatment could significantly alleviate inflammatory injury, revealing that
IL-17A is a critical requirement for early protection of infection. We also demonstrated that IL-17A was predominantly produced by
CD11b+Ly6G+ neutrophils. Although IL-17A could not significantly affect the antimicrobial responses of neutrophils, it could target the
proinflammatory macrophage (M1) programming and potentiate the M1’s defense against pneumonic plague. Mechanistically, IFN-g
treatment or IFN-g–activated M1 macrophage transfer could significantly mitigate the aggravated infection of IL-17A2/2 mice. Finally,
we showed that IL-17A and IFN-g could synergistically promote macrophage anti-infection immunity. Thus, our findings identify
a previously unrecognized function of IL-17A as an intrinsic regulator in coordinating neutrophil and macrophage antimicrobial
activity to provide protection against acute pneumonic plague. The Journal of Immunology, 2014, 192: 704–713.
The Journal of Immunology
705
neutrophils is required for IFN-g–activated classical M1 proinflammatory macrophage programming after Y. pestis challenge.
Intracellular staining and flow cytometry assays
Materials and Methods
Mice
2/2
were plated on Hottinger agar plates and incubated at 26˚C for 48 h.
Bacterial CFUs were then counted.
2/2
IL-17A
mice (20, 21) (B6 background) and IL-17RA
mice (22,
23) (B6 background) were obtained from The Jackson Laboratory (Bar
Harbor, ME). IFN-g2/2 mice (24) (B6 background) were obtained from
the Center of Model Animal Research at Nanjing University (Nanjing,
China). Rag12/2 mice, CD45.1+ mice, and CD45.2+ C57BL/6 mice were
obtained from Beijing University Experimental Animal Center (Beijing,
China). IL-17A2/2Rag12/2 double knockout (KO) mice were generated
by crossing Rag12/2 and IL-17A2/2 mice. All of these mice were bred
and maintained under specific pathogen–free conditions. Sex-matched
littermate mice aged 6–8 wk were mainly used for the experiments.
All of the animal experiments were performed in accordance with the
approval of the Animal Ethics Committee of the Beijing Institute of
Microbiology and Epidemiology, Beijing, China.
Bacteria
mAbs used for flow cytometry and neutralization
The cells were stained with Abs in PBS containing 0.1% (wt/vol) BSA and
0.1% NaN3 to analyze surface markers by flow cytometry. The following
Abs were purchased from different manufacturers: anti-CD11b (M1/70),
anti-F4/80 (BM8), anti–Gr-1 (RB6-8C5), anti-CD45.1 (A20), and antiCD45.2 (104; eBioscience, San Diego, CA); anti-CD11b (M1/70), antiCD45 (TU116), and anti-CD11c (HL3; BD Biosciences, San Diego, CA);
anti-CD45 (30-F11; BioLegend, San Diego, CA); anti-CXCR2 (clone
242216; R&D Systems, Minneapolis, MN); and anti-CD3 (145-2C11),
anti-CD19 (6D5), and anti–Gr-1 (RB6-8C5; Miltenyi Biotec, Bergisch
Gladbach, Germany). Anti–IL-17A mAb (clone 50104, R&D Systems;
IgG2a) was used to neutralize IL-17A in vivo.
Preparation of bronchoalveolar lavage fluid and lung
single-cell suspensions
Bronchoalveolar lavage fluid (BALF) was collected and harvested as
previously described (17). In brief, the trachea was exposed by producing
a midline incision and then cannulated with a sterile 22-gauge Abbocath-T
catheter. The bilateral BALF was collected after two 0.5-ml aliquots of
sterile saline were instilled. Approximately 0.9–1.0 ml of BALF was retrieved from each mouse. To prepare single-cell suspensions from lung
tissues, the lungs were perfused with saline containing heparin, minced,
and digested with collagenase and DNase I.
Isolation of neutrophils from bone marrow cells
Bone marrow was obtained as described before (16). Isolation of neutrophils from bone marrow cells was accomplished according to our previously published procedures (16, 30). Bone marrow CD11b+ cells were
isolated using anti-CD11b magnetic beads and positive selection columns
(Miltenyi Biotec). Ly6G+ cells were isolated using anti-Ly6G–PE mAb
(1A8; eBioscience) and positive immunomagnetic separation using a selection kit (Stem Cell Technologies, Vancouver, BC, Canada) or CD11b+
Ly6G+F4/80- neutrophils sorted on a FACSAria II (Becton Dickinson,
Franklin Lakes, NJ). Flow cytometry verified that all isolated cell purity
and viability was . 95%.
Measurement of bacterial burden
On the indicated days after challenge infection, BALF, lungs, and livers
were harvested and homogenized in saline. Serial dilutions of homogenates
Quantitative RT-PCR
RNA was extracted using an RNeasy kit (QIAGEN, Germany), and cDNA
was synthesized using SuperScript III reverse transcriptase (Invitrogen,
Carlsbad, CA). A LightCycler 480 (Roche, Switzerland) real-time PCR
system and primer and probe sets from Applied Biosystems were used for
quantitative PCR. The PCR primer sequences used in this study are
presented in Table I. To determine the relative expression of cytokine
mRNA in response to various conditions, the mRNA expression level of
each gene was normalized to the expression level of GAPDH, a housekeeping gene.
Quantification of cytokine by ELISA
Mouse BALF IL-17A levels were calculated from a standard curve obtained
from ELISA analysis of recombinant IL-17A (Bender MedSystems, Burlingame, CA).
Analysis of neutrophil degranulation
Neutrophils (2 3 106 cells per milliliter to 3 3 106 cells per milliliter) were
plated on wells in the presence or absence of 100 ng/ml TNF-a, as described previously, with slight modifications (31). The neutrophils were
incubated for 1 h at 37˚C; afterward, the cells were centrifuged and the
supernatants were removed to determine the release of elastase and
gelatinase. Elastase and gelatinase were determined to analyze enzyme
release in the supernatant after the neutrophils were degranulated using an
EnzChek Elastase Assay Kit and a Gelatinase Assay Kit (Invitrogen)
according to the manufacturer’s instructions.
Bone marrow transplantation models
A total of 5 3 106 donor CD45.2+ bone marrow cells from IL-17A2/2 or
CD45.1 WT littermates were transplanted i.v. in 8-wk-old CD45.1+ mice
that were lethally irradiated with 11 Gy to set up the complete chimeras, as
described previously (32, 33).
In vitro bactericidal activity by neutrophils and macrophages
Lung neutrophils and macrophages were sorted as described previously
(34, 35). Y. pestis strains (Y. pestis–GFP) were grown overnight at 26˚C.
These strains were subsequently washed in PBS and counted. The Y. pestis
containing a bacterial concentration of 106 CFU was incubated with 1 3
105 neutrophils in flat-bottom 96-well plates and in 200 ml RPMI medium
at 37˚C in 5% CO2 for 3 h. For phagocytosis experiments, neutrophils or
macrophages in some wells were collected at 30 min postinfection. The
neutrophils or macrophages were blocked with an anti-mouse FcgR mAb
(clone 2.4G2) and stained with anti-Ly6G–PE or anti-F4/80–PE. After
washing with cold PBS three times, the phagocytosis percentages of the
gated Ly6G+ cells or F4/80+ cells were then determined by an FACS scan.
The surviving Y. pestis CFUs were determined as previously described
(36), and the survival percentage was calculated.
Macrophage deletion
The Clophosome (FormuMax, Palo Alto, CA) was used for macrophage
deletion, as described previously (37). The dose was 0.15 ml for 20 g
animal body weight via the i.v. route before infection. The empty liposome
(Lipo) product was used as a negative control.
Oxidative burst assay
The respiratory burst was determined as previously described (38). Neutrophils isolated from the bone marrow were incubated in the presence of
1 mM dihydrorhodamine (Molecular Probes, Life Technologies, Grand
Island, NY) during stimulation with PMA (Sigma-Aldrich, St. Louis, MO).
The samples were incubated at 37˚C for 15 min before flow cytometry
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Y. pestis strain 91001 and strain 141 were used in this study. Strain 91001
belongs to a newly established Y. pestis biovar, Microtus (25), which is
considered avirulent in humans but highly lethal in mice (26). Strain 141 was
isolated from Marmota himalayana in the Qinghai-Tibet plateau and is highly
virulent to both humans and Rhesus macaques (27–29). Y. pestis bacilli were
grown overnight at 26˚C with continuous shaking in Bacto heart infusion
broth. After dilution to an OD of 0.1 at 620 nm, they were continuously
grown for 3–4 h at 26˚C, and then collected by centrifugation, washed with
saline, and quantified by OD measurement. The number of bacteria in the
inoculating dose was confirmed by plating. The LD50 of strain 91001 and
strain 141 via the intranasal route is ∼ 6 3 103 CFU and 1.6 3 104
CFU, respectively. Mice were infected intranasally with 0.5 LD50 to
generate survival curves, and 100 LD50 was permitted to assess lung pathological changes and other parameters following Y. pestis strain challenge.
Intracellular IL-17A (eBio17B7; eBioscience) and IL-17F (eBio18F10;
eBioscience) were analyzed by flow cytometry according to the manufacturer’s instructions. Flow cytometry data were acquired on an FACSCalibur (Becton Dickinson), and these data were analyzed with FlowJo
(TreeStar, San Carlos, CA). The number of cells of various populations
was calculated by multiplying the total number of cells by the percentages of each individual population from the same mouse, and the
result was averaged.
706
analysis. The neutrophils were identified by staining with CD11b and
Ly6G Ab.
NO production assay
Equal volumes of the culture supernatant or serum (100 ml) with Griess
reagent were incubated, and the absorbance at 550 nm was determined
using a microplate reader (Bio-Rad), as described previously (39).
Histopathology and immunohistochemistry
At 2 d postinfection, the entire lungs were excised and washed. The lungs
were fixed in 4% paraformaldehyde, embedded in paraffin, cut in 5-mm
sections, and stained with H&E for histopathological examination.
For immunohistochemistry, formalin-fixed paraffin-embedded tissues
were cut in 4-mm sections, and slides were stained to determine the neutrophils based on an immunoperoxidase method using a rat anti-mouse
Gr-1 mAb (BD Biosciences).
Statistical analysis
Results
IL-17A is a protective cytokine against pneumonic plague
We initially investigated the effects of IL-17A on the pneumonic
plague. IL-17A KO and wild-type (WT) mice were challenged in-
tranasally with Y. pestis strain 141 or 91001, respectively, to induce
the classical pneumonic plague as reported previously (2). As shown
in Fig. 1, IL-17A KO mice displayed symptoms of severe infectious
inflammation, whereas WT mice showed a delayed onset and markedly milder course of disease (Fig. 1A–C and data not shown). This
result was supported by a significantly lower percentage of survival,
higher body weight loss, and higher ratio between the lung and body
weight of the IL-17A KO mice. The WT littermates showed alleviation in the symptoms of infection. The number of Y. pestis after the
challenge showed that the lung and liver had higher bacterial CFUs in
the IL-17A KO mice than in the WT controls (Fig. 1D and data not
shown). Macroscopic and histological observation revealed a much
more severe pathological inflammation in the lungs of IL-17A KO
mice (Fig. 1E). These results indicated the possible role of IL-17A
signals in inducing acute pneumonic plague inflammation in mice.
IL-17A mRNA and IL-17 protein levels of the WT mice
gradually increased at different time points postinfection and
peaked at 24–48 h postinfection with Y. pestis strain 141 or
91001, but their levels subsequently began to decrease (Fig. 1F
and data not shown). This result is consistent with the relatively
milder symptoms of infection in the WT mice compared with
those in the IL-17A KO mice (Fig. 1A–E), and further indicated
that IL-17A may play a protective role in the early stage of the
pneumonic plague. Moreover, the IL-17A KO mouse, challenged
by Y. pestis strain 141 or 91001, showed a tendency similar to that
of the WT mouse with pneumonic plague. Because strain 91001 is
FIGURE 1. IL-17A is a protective cytokine against pneumonic plague. IL-17A KO mice were challenged with Y. pestis strain 141 and 91001 to cause
pneumonic plague. The percentage of survival of mice infected by Y. pestis strain 141 (A) or Y. pestis strain 91001 (B) was significantly reduced in IL-17A
KO mice compared with that of the WT littermates (**p , 0.01 and ***p , 0.001, log-rank test; n = 8–20 mice per group; data pooled from three
independent experiments), but the ratios between lung and body (C) were significantly increased in the IL-17A KO mice. Bacterial CFU of the lung infected
by Y. pestis strain 141 or 91001 was summarized at different time points after the indicated Y. pestis strain infection (D). Representative results are shown
from one of two or three independent experiments with similar results (statistical significance was measured by the Student t test. **p , 0.01, ***p ,
0.001). Infected mice developed severe pneumonic plague infection, as shown by the histological image, H&E sections of lung paraffin sections postinfection. The IL-17A KO mice exhibited severe tissue damage and massive inflammatory infiltration (E). The level of the IL-17A protein was analyzed by
ELISA (F) at the indicated time points challenged by Y. pestis strain 141 or 91001 in mice. (G) At 48 h postinfection in IL-17A KO, IL-17RA KO, and WT
mice, the lung bacterial CFUs were summarized. WT mice were treated with rmIL-17A and IgG2a isotypes or a rat anti-mouse IL-17A mAb prior to
infection. The lung bacterial CFUs were also summarized at the same time point. Statistical significance was measured by one-way ANOVA (p , 0.0001
for the overall ANOVA) with the Bonferroni multiple comparison test. ***p , 0.001 compared with WT group or the indicated groups. Representative
results are based on one of three independent experiments performed.
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Data are presented as mean 6 SD. Statistical analyses were performed
using the GraphPad Prism 5. A log-rank test was used for all the survival
data. The Student unpaired t test was used to compare the means between
two groups. For multiple comparisons, one-way ANOVA was used as indicated. A p value , 0.05 was considered statistically significant.
IL-17A PROTECTS AGAINST PNEUMONIC PLAGUE
The Journal of Immunology
less virulent to humans, and is convenient to use, this strain was part
of the following study. We hypothesized that acute bacterial infection induces IL-17A production via IL-17R, its receptor, to provide
protection against lung injury in mice with pneumonic plague. The
exaggerated effects were confirmed by more severe lung disease
and higher numbers of bacteria in the lungs of IL-17A 2/2 and
IL-17RA2/2 mice, compared with in the lungs of WT mice,
which demonstrated that the IL-17 signaling pathway was involved in the early stage of the pneumonic plague in mice (Fig.
1E, 1G). The exaggerated effects were also observed in WT
mice pretreated with neutralization of IL-17A with anti–IL-17A
mAb, but the protective effects were observed in WT mice
pretreated with IL-17A (Fig. 1G).
Neutrophils are the major sources of IL-17A in pneumonic
plague
and more severe pathological damage in the IL-17AKO→WT mice,
which lacked IL-17A in bone marrow cells. The same result was not
observed in WT→WT mice (Fig. 2B–D). Only IL-17AKO→IL17AKO mice showed more severe pathological changes and weight
loss compared with WT→IL-17A KO mice (Fig. 2D and data not
shown) in the complete bone marrow chimeras. Therefore, IL-17A
produced by bone marrow–derived cells contributed to protection
against pneumonic plague in mice.
We then generated IL-17A-Rag1DKO→Rag1KO and Rag1KO→Rag1KO bone marrow chimeras. IL-17A-Rag1DKO→Rag1KO
mice revealed a significantly higher percentage of deaths and bacterial
colony numbers compared with the Rag1KO→Rag1KO mice (Fig.
2E, 2F). Therefore, IL-17A produced from non-T and non-B bone
marrow–derived cells produced protection against pneumonic plague
in mice. T and B cell–deficient Rag1 KO mice were pretreated with
anti–IL-17A before being challenged with Y. pestis. We found that
these mice exhibited severe pathological inflammatory damage
(Supplemental Fig. 2A), lower percentage of survival (Supplemental
Fig. 2B), and higher bacterial colony number (Supplemental Fig. 2C),
similar to WT mice pretreated with anti–IL-17A mAb. These in vivo
studies suggested that cells other than T, B, NKT, and gdT cells
produced IL-17A at an early stage of pneumonic plague in mice.
The FACS results showed that IL-17A was secreted from the
recruited neutrophils (Fig. 3A, 3B), but not from T or NKT cells
(data not shown). A significant increase in IL-17A–producing
neutrophils was found at 48 h postinfection in the lungs (Fig. 3C).
To demonstrate the functional significance of IL-17A production
by neutrophils in pneumonic plague, we transferred the neutrophils from the bone marrow of WT (WT Neu→IL-17A KO) or
IL-17A KO mice (IL-17A KO Neu→IL-17A KO) to the recipient IL17A KO mice. The percentage of survival, pathological damage, and
number of bacterial colonies were reconstituted in the WT Neu→IL17A KO mice, but not in the IL-17A KO Neu→IL-17A KO mice
(Fig. 3D–F). Furthermore, the reconstituted protection in the WT
FIGURE 2. IL-17A produced from non-T and non-B bone marrow–derived cells protecting against pneumonic plague. (A) Lung CD45+ cells were
isolated from the lungs of infected mice at different time points. IL-17A expression was analyzed through intracellular staining with FACS. The data depict
the mean and SD of four mice per group. Statistical significance was measured by one-way ANOVA (p , 0.0001 for the overall ANOVA) with the
Bonferroni multiple comparison test. Results are shown for 0 h versus different hours postinfection, ***p , 0.001. WT BMC→WT and IL-17AKO
BMC→WT complete chimera mice were constructed. The mouse survival ratio infected by Y. pestis is shown in (B) (**p , 0.01, log-rank test; n = 12 mice
per group). Lung bacterial CFU (C) and histological H&E staining photo (D) at 48 h postinfection by Y. pestis. The percentage of survival (E) and lung
bacterial CFU (F) were investigated in Rag1KO BMC→Rag1KO and IL-17A2/2Rag12/2→Rag1KO complete chimera mice infected with Y. pestis at
different time points. Log-rank test for survival of mice, n = 7 mice per group, ***p , 0.001; Student t test for bacterial count, n = 4–5 mice per group;
representative results are shown from one of three independent experiments with similar results, ***p , 0.001.
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We performed a series of experiments to identify the cells that produce
IL-17A in lungs infected with Y. pestis. At 24 h postinfection, CD11b+
Ly6G+ neutrophils and F4/80+ macrophage infiltration were significantly increased over what was found in uninfected groups, as
revealed by immunohistological staining (Supplemental Fig. 1A) and
flow cytometry (Supplemental Fig. 1B–D). However, we could not
find any significant difference in the absolute number of infiltrating
immune cells (CD11b+Ly6G+ neutrophils, F4/80+ macrophages,
CD3+ T cells, and CD19+ B cells) between WT and IL-17A KO mice
challenged with Y. pestis (Supplemental Fig. 1). Importantly, we also
found that the IL-17A–producing CD45+ cells were gradually increased and peaked at 24 to 48 h postinfection, but these cells subsequently began to decrease (Fig. 2A). However, the same result was
not observed in IL-17F (data not shown). IL-17AKO→WT and
WT→WT complete bone marrow chimeras were generated to examine the contribution of bone marrow–derived cells as the source of
IL-17A in pneumonic plague. A higher percentage of deaths significantly correlated with higher bacterial colony numbers in the lungs
707
708
IL-17A PROTECTS AGAINST PNEUMONIC PLAGUE
Neu→IL-17A KO mice was blocked with 100 mg of anti-mouse IL17A mAb (Fig. 3D–F). These data demonstrated that neutrophils, and
not T, NKT, gdT, or B cells, are the major source of IL-17A production that protects against pneumonic plague in mice.
IL-17A signaling is not necessary in neutrophil bactericidal
activities that provide protection against pneumonic plague
Proinflammatory cytokines mediate bacterial lung inflammation.
For this reason, we investigated the time course changes of the
cytokine expression in lung samples from the WT mice by realtime PCR (Table I). Postinfection, IL-6, TNF-a, IL-1b, CXCL1,
and CXCL2 gradually increased and peaked at 24–48 h, but these
cytokines subsequently started to decrease (Fig. 4A). Although
IL-6 and TNF-a were decreased in pneumonic plague in IL-17A
KO and IL-17R KO mice, compared with WT mice, IL-17 signaling could not significantly affect neutrophil bactericidal activities
Table I.
(Fig. 4B). Reactive oxygen metabolites are generated after neutrophil activation and are important to eradicate microorganisms. Bone
marrow neutrophils from IL-17A–deficient mice challenged with
Y. pestis showed a similar level of oxidative burst and peaked 20 min
after these neutrophils were stimulated with PMA, compared with
those from the WT controls (Fig. 4C). The mRNA expression of
granule proteins such as elastase and gelatinase did not significantly
differ in the neutrophils of the IL-17A KO mice compared with those
of the WT mice (Fig. 4D). These results indicated that IL-17A is
unlikely to be necessary in regulating neutrophil function to protect
against pneumonic plague. Furthermore, we did not observe significant differences between IL-17A KO and WT mice in the abilities of
neutrophils to phagocytose and eradicate Y. pestis in vitro. At 1 h
postinfection, a large number of Y. pestis were phagocytosed by
neutrophils, but no significant differences in the phagocytosis percentages of Y. pestis were observed between IL-17A KO and WT mice
Primer sequences used in this study
Genes
Arginase I
CXCL1
CXCL2
iNOS
IL-1b
IL-6
IL-17A
TNF-a
GAPDH
Forward Primer (5ʹ to 3ʹ)
CCA
GCA
GCC
CAC
TGG
GCA
CTC
GAG
GAC
GAA
CCC
CAG
CAA
GAA
ATG
AGA
TGA
TTC
GAA TGG
AAA CCG
ACA GAA
GCT GAA
ACA ACA
GCA ATT
CT ACCT
CAA GCC
AAC AGC
AAG
AAG
GTC
CTT
GTG
CTG
CAA
TGT
AAC
AGT
TCA
ATA
GAG
GTC
ATT
CCG
AGC
TCC
CAG TGT
TAG
GCC
CG
AGG
GTA TG
TTC C
C
CAC
Reverse Primer (5ʹ to 3ʹ)
GCA
AGA
CTC
CGT
CCA
AAG
ATG
CTC
TCC
GAT
AGC
CTC
GGC
TCA
GAC
TGG
CTG
ACC
ATG
CAG
CTT
TTT
GAG
TCT
TGG
GTA
ACC
CAG
CGT
TCC
GGG
GCA
GGC
TCC
TGA
CTG
GGA
TCA
AGG
CTC
AGG
TTT
AGC
GAT
TTG
GTC
CCA
TCA
CTC
AGG
GTC
TTT
AGC
CTG
ACC
GA
GTT A
AA
TTT CT
CC
AAA
TA
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FIGURE 3. IL-17A produced by neutrophils responsible for protecting against the pneumonic plague. (A) Expression of IL-17A in Ly6G+ neutrophils
gated from the CD45+ cell in the lungs of infected mice at different time points with FACS. The percentage changes are summarized in (B), and the absolute
IL-17A+Ly6G+ cell number is calculated in (C). The data depict the mean and SD of four mice per group, and statistical significance was measured by oneway ANOVA (p , 0.0001 for the overall ANOVA) with the Bonferroni multiple comparison test. ***p , 0.001 compared with 0 h. (D) WT, not IL-17A
KO, bone marrow–derived neutrophils transferred to IL-17A KO mice could mitigate the severe inflammatory injuries challenged with Y. pestis. However,
in one group (WT PMNs→IL-17A KO mice + anti-IL-17A) 100 mg anti–IL-17A mAb was administrated by i.v. injection, which could not mitigate the
severe inflammatory injuries infected with Y. pestis. The percentage of survival of infected mice was summarized. ***p , 0.001, log-rank test; n = 9–12
mice per group. The representative histological H&E staining image is shown in (E), and the lung bacterial CFU is summarized in (F). Statistical significance was measured by one-way ANOVA (p , 0.0001 for the overall ANOVA) with the Bonferroni multiple comparison test. Representative results are
based on one of two or three independent experiments performed with similar results.
The Journal of Immunology
709
(Fig. 4E). Moreover, no significant differences in the release of
gelatinase and elastase in the neutrophils were observed between IL17A KO and WT mice (Fig. 4F). The survival percentage of Y. pestis
in the IL-17A KO mice could be comparable to that of the WT
controls (Fig. 4G). All of these results indicated that neutrophilderived IL-17A could not affect neutrophil bactericidal activities in
protecting against pneumonic plague in mice.
IL-17A that targets IFN-g–activated M1 macrophages provides
protection against pneumonic plague in mice
IFN-g is an important cytokine that stimulates classically activated macrophages (M1), which figure greatly in providing protection against pneumonic plague infection (40, 41). However, the
macrophage-mediated immune response might be affected by
IL-17 signaling. To test this hypothesis, we observed the inflammatory effects of pneumonic plague in IFN-g signaling deficiency.
Bacterial colony number (Fig. 5A) and pathological damage (data not
shown) were significantly aggravated at 24 h postinfection in mice
treated with a neutralized anti–IFN-g Ab, compared with WT mice
treated with an IgG1 isotype control. This result was not observed
in IL-17A KO mice. The increased number of bacterial colonies and
the aggravated pathological damage were also observed in IFN-g–
deficient mice (data not shown). These results further suggested that
the IFN-g pathway is involved in IL-17A–mediated protective effects
against pneumonic plague. However, how does IFN-g mediate this
protective effect? Macrophages have a major role in providing protection against pneumonic plague infection in early stages, and this
protective role of macrophages usually requires IFN-g signaling (42,
43). Thus, macrophages probably participated in providing protective
effects against pneumonic plague in mice.
To verify this hypothesis, we performed a macrophage-depletion
experiment in vivo (Fig. 5B, 5C). The number of bacterial colonies
and the pathological damage were significantly aggravated at 48 h
postinfection in the WT mice with depleted macrophages (treated
with clodronate [CL] and Lipo), but this result was not observed in
IL-17A KO and IFN-g KO mice (Fig. 5D and data not shown).
This result revealed that IL-17A– and IFNg–mediated protection
against pneumonic plague was related to the macrophage population in vivo. Our results are consistent with the possibility
that IL-17A– and IFN-g–activated macrophages mediated protection against pneumonic plague in mice, suggesting that these
pathways are very important in macrophage-mediated protection
against Y. pestis infection. Thus, IFN-g and IL-17A signaling
contributed to this process.
We treated the infected WT and IL-17A KO mice with murine
IFN-g to detect the interaction between the macrophages with
IFN-g and IL-17A pathways involved in providing protection
against pneumonic plague. The IL-17A KO mice received
recombinant murine (rm)IFN-g (3000 U per mouse) 1 h prior to
the bacterial challenge. The rmIFN-g could almost completely
provide protection against pneumonic plague in IL-17AKO mice,
as observed in the changes in bacterial CFU, but not in the mice
treated with CL and Lipo (Fig. 5E). These results are consistent
with the possibility that IL-17A protected the mice against
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FIGURE 4. IL-17A signaling is not necessary for neutrophil bactericidal activity in protecting against pneumonic plague. (A) The lung mRNA expression of IL-6, TNF-a, IL-1b, CXCL1, and CXCL2 was detected by quantitative real-time PCR in WT mice at different time points after Y. pestis
challenge. (B) IL-17A KO and IL-17RA KO showed reduced lung expressions of IL-6 and TNF-a infected by pneumonic plague at 24 h compared with the
WT control group. No significant difference in CXCL1 and CXCL2 was found among WT, IL-17A KO, and IL-17RA KO mice. Statistical significance was
measured by one-way ANOVA (p = 0.008 for the overall ANOVA) followed by the Tukey multiple comparison test. **p , 0.01 compared with the WT
group. Representative results are shown from one of two independent experiments with similar results. (C) Respiratory burst assayed by isolated bone
marrow neutrophils of the pneumonic plague mice was determined via the oxidation of dihydrorhodamine 123 after activation with 40 ng/ml of PMA. The
data represent the mean fluorescent intensity of all cells with a signal above the background and are normalized for the WT normal values (mean 6 SD;
n = 3). The data were obtained from one of four independent experiments with similar results. (D) The neutrophils were isolated from WT and IL-17A KO
mice infected by pneumonic plague at 24 h. The mRNA expressions of elastase and gelatinase were detected as described in Materials and Methods. (E–G)
The neutrophil defense response to Y. pestis infection was determined in vitro. The neutrophils isolated from WT and IL-17A KO mice were cultured with
Y. pestis–GFP. The percentage of phagocytosis of neutrophils was determined by FACS (E). The supernatant was collected, and the release of elastase and
gelatinase was detected as described in Materials and Methods (F). Bacterial survival in the culture was determined as described in Materials and Methods
(G), and the experiment was performed in triplicate. The representative results are shown based on one of three independent experiments performed.
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IL-17A PROTECTS AGAINST PNEUMONIC PLAGUE
pneumonic plague via IFN-g–activated M1 macrophages. The
aggravated number of bacterial colonies, percentage of survival,
and pathological changes could be significantly ameliorated in
mice when M1 bone marrow–derived macrophages activated by
LPS and IFN-g were transferred to IL-17A KO mice before being
subjected to Y. pestis challenge (Fig. 5G, Supplemental Fig. 3).
IL-17A/IL-17R signaling contributes to M1 macrophage
programming and protects from pneumonic plague
We analyzed the production of NO as well as arginase and inducible NO synthase (iNOS) expressions during the programming
of macrophages in vitro to investigate the function of IL-17A/IL17R signaling in programming M1 macrophages. As expected, NO
production (Fig. 6A), as well as iNOSmRNA (Fig. 6B) and protein
(Fig. 6C) expression, was significantly higher in the group treated
with LPS + IFN-g + IL-17 compared with the group treated with
LPS or LPS + IFN-g alone (p , 0.001). No significant differences
were found between arginase protein (Fig. 6D) and mRNA (Fig.
6E) expression in the combined treatment group (IL-17, IFN-g,
and LPS) compared with LPS alone or LPS + IFN-g–treated
groups, as detected by real-time PCR and ELISA.
Bactericidal capacity is the gold standard used to evaluate
macrophage function. We observed an increase in phagocytosis of
the macrophages treated with IFN-g and IL-17A. Y. pestis was
significantly reduced in in vitro coculture systems, compared with
IFN-g or IL-17A treatment alone. Macrophages were treated with
IL-17A or IFN-g or the two together or none for 2 h before infection. More Y. pestis were phagocytized in the cytokine com-
bined group than in that treated with IFN-g or IL-17A alone (Fig.
6F). The survival percentage of Y. pestis in macrophages treated
with IFN-g + IL-17A was lower than that in macrophages treated
with IFN-g or IL-17A alone (Fig. 6G). These results revealed that
IL-17A signaling contributed to IFN-g–activated M1 macrophage
protection against pneumonic plague.
The bactericidal capacity of the macrophages in vivo was investigated. Y. pestis–GFP (1 3 108 CFU) was injected in the
peritoneal cavity of mice. The phagocytosis percentage and the
survival of Y. pestis in macrophages were investigated as described
in Materials and Methods. We observed the decreased ability of
IL-17A KO macrophages to phagocytose and eradicate Y. pestis in
the in vivo system compared with that of the WT mice. At 12 h
postinfection, IL-17A KO macrophages phagocytosed Y. pestis to
a lesser extent than did those in the WT control mice (Supplemental Fig. 4A, 4B). The survival percentage of Y. pestis was
significantly increased in IL-17A KO mice compared with WT
control mice. Thus, IL-17A produced by neutrophils protected the
mice against pneumonic plague in an IFN-g–activated macrophage-dependent manner (Fig. 7).
Discussion
Plague is one of the deadliest infectious diseases (1, 41). The
causative agent, Y. pestis, is a Gram-negative facultative bacterium
that is naturally transmitted from rodents to humans by fleas (2).
Y. pestis bacilli typically infect the nearest skin-draining lymph
nodes, which swell to produce diagnostic buboes upon transmission by a flea bite (44). The bubonic form of plague often leads to
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FIGURE 5. IL-17A/IL-17R targeting in IFN-g–activating macrophages protects against pneumonic plague. (A) The pneumonic plague bacterial CFUs
were summarized from the IL-17A KO mice and the WT control mice or pretreated with isotype IgG1 or rat–anti-mouse IFN-g mAb. Statistical significance was measured by the Student t test. n = 3–5; ***p , 0.001. (B and C) Lung CD11b+F4/80+ macrophages were detected using PBS, PBS + Lipo, or
CL + Lipo. The macrophages were significantly decreased in the lungs after treatment with CL + Lipo. Data shown are mean and SD of eight mice pooled
from three independent experiments. Statistical significance was measured by one-way ANOVA (p , 0.0001 for the overall ANOVA) with the Bonferroni
multiple comparison test. ***p , 0.001 compared with Lipo control group. (D) WT, IL-17A KO, and IFN-g KO mouse macrophages were deleted using
CL and Lipo and then challenged with Y. pestis for 48 h. The lung bacterial CFUs were summarized. Statistical significance was measured by the Student t
test. n = 3–5; ***p , 0.001. Representative results are shown from one of two independent experiments with similar results. (E) The mIFN-g treatment can
rescue the infected IL-17AKO mouse bacterial CFU changes, but not in the macrophage-depleted mice treated with CL + Lipo. Statistical significance was
measured by the Student t test. n = 3–5; ***p , 0.001. Representative results are shown from one of two independent experiments with similar results. (F)
Bone marrow–derived macrophages preactivated by LPS and IFN-g transfer can repair pathological lung injury changes from IL-17A KO compared with
that from the WT control or in combination with unactivated macrophage control. The percentage of mouse survival is consistently summarized in (G)
(**p , 0.01, log-rank test; n = 8–10; data are pooled from two independent experiments).
The Journal of Immunology
711
sepsis and occasionally progresses to secondary pneumonic infection. Pneumonic plague is almost always lethal in humans (5).
Moreover, pneumonic plague can spread from person to person via
infectious respiratory droplets (5). Substantial concerns are increasing because Y. pestis could be exploited as a biological
weapon and extensively antibiotic-resistant Y. pestis strains are
currently emerging (45). The question remains whether some
natural intrinsic protective mechanisms are found in the pathological course of pneumonic plague. Numerous efforts have been
devoted to the development of plague vaccines, such as subunit
vaccines containing Y. pestis F1 and LcrV proteins that were recently used in human clinical trials (46, 47). In the current study,
IL-17A was considered a natural protective cytokine that critically
provides protection against early pneumonic plague infection by
linking the reciprocal sequencing modulation among neutrophils,
macrophage activation, and programming.
IL-17 has become a target molecule since its discovery, particularly after the Th17 lineage has been discovered and has become one of the most studied cytokines in immunology (48). Th17
cells account for only a fraction of IL-17 produced in vivo. IL-17
is mostly derived from innate lymphoid populations of neutrophils, monocytes, NK cells, and lymphoid tissue inducer-like
cells and can rapidly produce IL-17A and IL-17F, particularly at
mucosal sites (48–50). Emerging evidence has indicated that IL17 is mainly protective at the lung surface, where IL-17 has
a nonredundant function in controlling infection (18). Mice defective in IL-17 signaling are highly susceptible to infection by
Klebsiella pneumoniae, whereas IL-4, IL-12, and IFN-g are not
necessary in the protection against such an infection (51, 52). IL-17
also controls responses to fungal pathogens because mice that lack
IL-23 and receptors such as IL-17RA and IL-17RC are susceptible to
infection with Candida albicans (53). Recent studies (54) showed
that prime vaccination with D27-pLpxL confers better protection
than prime-only vaccination, and the prime also increases pulmonary
numbers of Th17. This finding suggests that IL-17 contributes to cellmediated defense against pulmonary Y. pestis infection. However, the
exact innate immediate protective mechanism, especially during
early acute infectious disease, remains unclear. In our study, we
initially clarified that IL-17 also exhibited a protective role in infectious disease. We demonstrated that the deficiency in IL-17A or
IL-17R and mAb neutralization of IL-17A aggravated the acute
pneumonic plague infection. IL-17A produced by Ly6G+ neutrophils,
not by NKT or gdT cells, was important in providing protection
against early pneumonic plague infection in mice (Figs. 2 and 3 and
data not shown). This result confirmed that IL-17A, a natural cytokine
produced by neutrophils, provided protection in early acute pneumonic plague infection.
Furthermore, our results showed that IL-17 could not significantly change neutrophil bactericidal activities (Fig. 4D–G), although IL-17A deficiency could significantly decrease the production of IL-6, TNF-a, and IL-1b (Fig. 4A, 4B). The results further
revealed that the neutrophil-derived IL-17A could not affect neutrophil bactericidal activities that provide protection against pneumonic plague in mice.
Therapeutic administration of the immune serum from convalescent mice protects naive WT mice against lethal pulmonary
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FIGURE 6. IL-17A signaling promotes IFN-g–activated M1 macrophage programming and function. (A) Significantly higher NO levels of macrophages
were found in the combined treated groups with rmIL-17A, LPS, and rmIFN-g compared with LPS or LPS + rmIFN-g treatment. More iNOS mRNA (B),
protein (C), and macrophage expression in the combined treated groups with rmIL-17A, LPS, and rmIFN-g compared with LPS or LPS + rmIFN-g
treatment. Statistical significance was measured by one-way ANOVA (p , 0.0001 for the overall ANOVA) followed by the Bonferroni multiple comparison
test. *p , 0.05, ***p , 0.001 compared with the indicated group; representative results are shown from one of three independent experiments with the
similar results. The arginase level (D) and arginase I (E) mRNA expressions of the macrophages were determined in the combined treatment groups with
rmIL-17A, LPS, and rmIFN-g, LPS alone, or LPS + rmIFN-g treatment. (F) Combined rmIL-17A and rmIFN-g treatment can potentiate the macrophage
phagocytosis of Y. pestis, compared with rmIL-17A or rmIFN-g treatment. (G) Combined rmIL-17A and rmIFN-g treatment can decrease the survival
percentage of Y. pestis compared with rmIL-17A or rmIFN-g treatment. Representative results are shown from two (D and E) or three (F and G) independent
experiments performed with similar results. Statistical significance was measured by one-way ANOVA (p , 0.0001 for the overall ANOVA) followed by
the Bonferroni multiple comparison test. **p , 0.01, ***p , 0.001 compared with the indicated groups.
712
IL-17A PROTECTS AGAINST PNEUMONIC PLAGUE
and chemokines are produced by airway epithelial and endothelial
cells to promote the recruitment and activation of neutrophils and to
secrete IL-17A in response to plague. IL-17 signaling subsequently
occurs and strengthens IFN-g–activated M1 macrophage programming and function (Fig. 7). Thus, our study demonstrated that
IL-17A coordinated neutrophils and macrophages, which provide protection against early pneumonic plague infection.
Acknowledgments
We thank Dr. Hui Yang for review of the manuscript and Zhizhen Qi for help
in animal observation.
Disclosures
The authors have no financial conflicts of interests.
References
Y. pestis challenge (55). This immune serum also poorly protects
gene-targeted mice that lack the capacity to produce TNF-a or
IFN-g, suggesting that TNF-a and IFN-g contribute to serum
therapy–mediated protection against pneumonic plague. However,
acute regulatory mechanisms by which TNF-a and IFN-g contribute to protect the cells against pneumonic plague remains
unclear. In this study, gene-deficient mice or a macrophage-depleted
system demonstrated that IFN-g is necessary for macrophageactivated protection against pneumonic plague, using a neutralized
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Neutrophils immediately respond as innate immune cells during
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promote inflammation by increasing proinflammatory cytokines and chemokines, including CXCL1 and CXCL2. CXCL1/2-mediated neutrophil
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