Type I IFN Signaling Is Crucial for Host
Resistance against Different Species of
Pathogenic Bacteria
This information is current as
of June 18, 2017.
Giuseppe Mancuso, Angelina Midiri, Carmelo Biondo,
Concetta Beninati, Sebastiana Zummo, Roberta Galbo,
Francesco Tomasello, Maria Gambuzza, Giancarlo Macrì,
Alessia Ruggeri, Tomas Leanderson and Giuseppe Teti
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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 © 2007 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2007; 178:3126-3133; ;
doi: 10.4049/jimmunol.178.5.3126
http://www.jimmunol.org/content/178/5/3126
The Journal of Immunology
Type I IFN Signaling Is Crucial for Host Resistance against
Different Species of Pathogenic Bacteria1
Giuseppe Mancuso,* Angelina Midiri,* Carmelo Biondo,* Concetta Beninati,*
Sebastiana Zummo,* Roberta Galbo,* Francesco Tomasello,* Maria Gambuzza,*
Giancarlo Macrı̀,* Alessia Ruggeri,* Tomas Leanderson,† and Giuseppe Teti2*
T
he IFNs were the first cytokines discovered, having been
described nearly 50 years ago as products of virus-infected cells capable of inducing a virus-resistant state (1).
IFNs are classified into type I and II based on receptor usage,
molecular structure, and sequence homology (2, 3). Type I IFNs,
which comprise multiple IFN-␣ and single IFN-␤ and IFN-␻ subtypes, bind to a common, ubiquitously expressed heterodimeric
receptor (IFN-␣␤ receptor or IFNAR),3 composed of IFNAR1 and
IFNAR2 subunits. Type II IFN, or IFN-␥, signals through a different receptor composed of two IFN-GR1 and two IFN-GR2
chains. Together with few other mediators, including TNF-␣ and
IL-12, type I IFNs are considered as primary cytokines that are
produced directly in response to microbial products and are therefore likely to shape downstream events in innate and adaptative
immune responses (2, 4).
The two IFN types have distinct, nonredundant activities and
may have evolved to complement each other as innate host defense
factors. Each IFN type regulates hundreds of partially overlapping
secondary genes, the products of which orchestrate a comprehensive antimicrobial program (2– 4). Although endowed with a more
limited range of antiviral activities than type I IFNs, IFN-␥ can
potently activate macrophages to produce proinflammatory cyto*Dipartimento di Patologia e Microbiologia Sperimentale, Università degli Studi di
Messina, Messina, Italy; and †Immunology Group, University of Lund, Lund, Sweden
Received for publication July 26, 2006. Accepted for publication December 8, 2006.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported in part by a Progetto di Ricerca di Intgresse Nationale
grant from Italian Ministry of University and Research of Italy (2004068345_004).
2
Address correspondence and reprint requests to Dr. Giuseppe Teti, Dipartimento di
Patologia e Microbiologia Sperimentale, Policlinico Universitario, Via Consolare
Valeria, 1.98125 Messina, Italy. E-mail address: [email protected]
Abbreviations used in this paper: IFNAR, IFN-␣␤ receptor; GBS, group B streptococci; KO, knockout; WT, wild type.
3
Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00
www.jimmunol.org
kines and to kill ingested nonviral organisms, including bacterial,
fungal, and protozoan pathogens (3). Conversely, type I IFN has
been traditionally assigned a minor role in antibacterial host defenses (5). Recently, however, conserved bacterial products, including LPS and prokaryotic DNA, were found to activate distinctive signal transduction pathways that merge, after pathogen
recognition, with those activated by viruses and lead to high-level
type I IFN production (6).
Whether type I IFN has a role in antibacterial defenses is presently unclear. Contrasting results have been obtained according to
the pathogen species and experimental model (e.g., in vivo vs in
vitro). In in vitro models, treatment of macrophages or fibroblasts
with type I IFNs generally limited intracellular replication of bacteria, including Chlamydia, Legionella, and Shigella spp (7–10).
However, the addition of IFN-␣ or IFN-␤ to human macrophages
increased the intracellular replication of Mycobacterium bovis
(11). In in vivo mouse models, administration of type I IFN exacerbated pulmonary tuberculosis (12), but had protective effects
in infections by Salmonella typhimurium (13) or Listeria monocytogenes (14).
Very little is known of the physiologic role of endogenously
produced, as opposed to exogenously added, type I IFN in the
context of bacterial infections. The effects of type I IFN signaling
have been studied recently in IFN-␣␤R-deficient mice infected
with the facultative intracellular pathogens Mycobacterium tuberculosis or L. monocytogenes. There were only slight differences
between IFN-␣␤R-deficient and wild-type (WT) mice in lung CFU
after aerosol challenge with M. tuberculosis (15). Surprisingly,
three recent studies indicate that type I IFN signaling may actually
decrease host resistance against L. monocytogenes infection (16 –
18). The mechanisms underlying these detrimental effects are presently unknown, but may be related to the proapoptotic effects of
Listeria-induced type I IFN (17–19).
It is reasonable to assume that at least a portion of the antiviral
program triggered by IFN-␣␤ is ineffective against bacteria, owing
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It is known that host cells can produce type I IFNs (IFN-␣␤) after exposure to conserved bacterial products, but the functional
consequences of such responses on the outcome of bacterial infections are incompletely understood. We show in this study that IFN-␣␤
signaling is crucial for host defenses against different bacteria, including group B streptococci (GBS), pneumococci, and Escherichia coli.
In response to GBS challenge, most mice lacking either the IFN-␣␤R or IFN-␤ died from unrestrained bacteremia, whereas all wild-type
controls survived. The effect of IFN-␣␤R deficiency was marked, with mortality surpassing that seen in IFN-␥R-deficient mice. Animals
lacking both IFN-␣␤R and IFN-␥R displayed additive lethality, suggesting that the two IFN types have complementary and nonredundant roles in host defenses. Increased production of IFN-␣␤ was detected in macrophages after exposure to GBS. Moreover, in the
absence of IFN-␣␤ signaling, a marked reduction in macrophage production of IFN-␥, NO, and TNF-␣ was observed after stimulation
with live bacteria or with purified LPS. Collectively, our data document a novel, fundamental function of IFN-␣␤ in boosting macrophage responses and host resistance against bacterial pathogens. These data may be useful to devise alternative strategies to treat
bacterial infections. The Journal of Immunology, 2007, 178: 3126 –3133.
The Journal of Immunology
3127
to fundamental differences between viruses and prokaryotic organisms (20 –22). In contrast, it is difficult to understand how the host
has evolved specific mechanisms to produce IFN-␣␤ in response
to bacteria, if such responses are useless or detrimental. To gain
further insights, it may be useful to assess the role of type I IFN in
defenses against pathogens differing in their relationship with host
cells. Because only intracellular pathogens have been studied to
date, the present study investigated the consequences of defective
type I IFN signaling during infection with extracellular pathogens,
including group B streptococci (GBS), Streptococcus pneumoniae,
and encapsulated Escherichia coli. These encapsulated bacteria
impose an enormous burden on human health. S. pneumoniae is
estimated to cause 10 million deaths annually in the world (23).
GBS and encapsulated E. coli are major causes of sepsis and meningitis in the newborn and are being increasingly associated with
invasive disease in the adult population (24). Moreover, several
signal transduction pathways contributing to innate resistance
against these bacteria have been elucidated recently (25–28).
We found in this study that type I IFN is a requirement for resistance against GBS, pneumococci, and E. coli. Increased susceptibility
to infection could be related to defective production of IFN-␥, NO,
and TNF-␣ by macrophages in the absence of IFN-␣␤ signaling.
Materials and Methods
Mice
IFN-␣␤R⫺/⫺, IFN-␥R⫺/⫺, IFN-␣␤, and IFN-␥R⫺/⫺ double knockout (KO)
and 129Sv/Ev control WT mice were purchased from B&K Universal.
IFN-␤⫺/⫺ mice were developed, as described (29). C57BL/6 mice (Charles
River Laboratories) were used as controls for the IFN-␤-deficient mice.
Mice used in the present study were housed under specific pathogen-free
conditions in enclosed filter top cages of the Department of Pathology and
Experimental Microbiology of the University of Messina. The mice were
fed clean food and water ad libitum. All of the procedures described in the
present study were in agreement with the European Union guidelines of
animal care and were approved by the relevant committees.
Reagents and bacterial strains
Chemicals were purchased from Sigma-Aldrich, unless indicated otherwise. E. coli O111:B4 ultrapure LPS was obtained from InvivoGen (distributed by Labogen). The following bacterial strains were used in the
present study: COH-1, a highly virulent type III GBS strain, provided by C.
Rubens (University of Washington, Seattle, WA); GBS ␤-h/c⫺ (30), a
hemolysin-defective mutant of COH-1, provided by V. Nizet (University
of California, San Diego, La Jolla, CA); strain 2603 V/R, a type V GBS
(31); L. monocytogenes 265, a previously described clinical isolate (32). S.
pneumoniae strain D39 and E. coli strain K1 E-R8 were provided, respectively, by M. Oggioni (University of Siena, Siena, Italy) and A. Caprioli
(Istituto Superiore di Sanità, Rome, Italy). All bacterial strains were grown
to the mid-log phase in Todd-Hewitt broth, washed three times in PBS
(0.01 M phosphate and 0.15 M NaCl (pH 7.2)), and diluted to the appropriate concentration. The number of viable bacteria used in each experiment was carefully determined by plate counting. A Limulus amebocyte
lysate test (Pyrotell assay; Associates of Cape Cod), conducted according
to the instruction of the manufacturers, revealed no detectable amounts of
LPS (i.e., ⱖ0.01 EU/ml) in any of the Gram-positive bacterial preparations.
Bacterial infection models
In GBS sepsis models, neonatal or adult mice of either sex were injected
locally (s.c. or i.p.) with viable bacteria. Under these conditions, GBS are
contained at the inoculation site or spread systemically, depending on the
bacterial dose and the intrinsic ability of the host to control microbial
growth (26, 33). Infection has a rapid course in these models, and the
animals either die of septic shock or clear the infection within 1– 4 days.
Mouse pups (24 – 48 h old) were infected s.c. with the type III strain
COH-1 in 30 ␮l of PBS. Adult (6-wk-old) mice were challenged i.p. with
strain 2603 V/R, belonging to a serotype (type V) that accounts for most of
adult human infections. Mice were observed daily for 14 days after infection, but deaths were never recorded after 4 days. In additional experiments, mice were killed at 18 or 36 h after challenge for the determination
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FIGURE 1. Effects of type I and II IFN
signaling on the outcome of GBS infection in
neonatal mice. A, Survival of WT (129Sv/
Ev), IFN-␣␤R⫺/⫺, IFN-␥R⫺/⫺, and double
KO (IFN-␣␤ and IFN-␥R⫺/⫺) neonatal mice
after s.c. challenge with 20 CFU of GBS
strain COH-1. Bacterial numbers in the
blood (B), kidney (C), plasma TNF-␣ (D),
and IL-6 (E) levels after a similar challenge
are also shown. Log CFU were expressed as
means ⫾ SDs of 10 determinations, each
conducted on a different animal. Cytokine
levels were expressed as means ⫾ SDs of
five determinations, each conducted on a different animal. ⴱ, p ⬍ 0.05 vs WT mice;
#, p ⬍ 0.05 vs IFN-␥R⫺/⫺ mice; $, p ⬍ 0.05
vs IFN-␣␤R⫺/⫺ mice. ND, Nondetectable.
IFN-␣␤ AND BACTERIAL INFECTIONS
3128
of blood and kidney CFU using blood agar. Because kidney weight did not
differ in different groups of mice, results were expressed as CFU/kidney.
In additional sepsis models, E. coli E-R8 and S. pneumoniae D39 were
injected i.v. in 6-wk-old mice of either sex. We also used a pneumococcal
meningitis model in which adult mice were inoculated with S. pneumoniae
D39 via an intracranial, subarachnoidal route, as previously described (34).
In this model, which mimics several features of naturally occurring bacterial meningitis, pneumococci replicate in the subarachnoidal space and
cause little or no encephalitis (34).
Culture and stimulation of peritoneal macrophages
Resident or thioglycolate-elicited mouse macrophages were obtained, as
described (35), and incubated with live bacteria at different multiplicities of
infection for 2 h. Subsequently, the plates were washed with warm medium
and cells were further cultured for various times in the presence of gentamicin (100 ␮g/ml) before harvesting cells and/or supernatants. In control
experiments, cells were cultured continuously in the presence of E. coli
LPS. In reconstitution experiments, rIFN-␤ (with a sp. act. of 2.42 ⫻ 107
U/mg; PBL Biomedical Laboratories, distributed by R&D Systems) was
added to macrophages from IFN-␤⫺/⫺ mice at the same time of addition of
the bacterial stimuli. After 2 h, the monolayers were washed and further
cultured in the presence of medium containing the recombinant cytokine
and gentamicin. Macrophage viability was checked at the end of the experiments and was always higher than 90%.
Cytokine and NO measurements
TNF-␣, IFN-␥, IFN-␤, and IL-6 concentrations were measured by ELISA
in culture supernatants or in the plasma of infected mice using the mouse
TNF-␣ module set (Bender MedSystems), the murine IFN-␥ reagent set
(Euroclone), the mouse IFN-␤ ELISA (PBL Biomedical Laboratories), and
the murine IL-6 reagent set (Euroclone). The lower detection limits of these
assays were 16, 25, 15.6, and 15.6 pg/ml, respectively. NO concentration
was determined by measuring nitrite formation, as described (36), using the
Griess reagent (1% sulfanilamide in 2.5% phosphoric acid-0.1% n-1-napthylethylenediamide dichlorique). After a 30-min incubation at room temperature with agitation, absorbance was measured at 540 nm. NO2⫺ was
quantified using NaNO2 as a standard.
IFN-␣␤ mRNA analysis by quantitative real-time PCR
IFN-␣4 and IFN-␤ mRNA were measured in peritoneal macrophages or in
the spleens of infected mice. Peritoneal macrophages (5 ⫻ 106) were collected after culture by scraping and vigorous pipetting in cold PBS, sedimented by centrifugation, and mixed with the RNeasy kit lysis buffer (Qiagen). Spleens, obtained immediately after sacrifice, were homogenized in
the same lysis buffer using the Tissue Lyser MM301 (Retsch; distributed
by Incotec Italia). mRNA was extracted with the RNeasy kit, per the manufacturer’s instructions.
For the quantification of IFN-␣4 and IFN-␤ mRNA, real-time quantitative RT-PCR assays were conducted, in duplicate, with an Applied Biosystems 7500 (Applied Biosystems), as described (37). PCR conditions
were as follows: 50°C, 30 min; 95°C, 10 min; (95°C, 15 s; 60°C, 1 min) ⫻
40 cycles. Real-time PCR data were normalized in each individual sample
by the level of ␤-actin expression. Gene expression was measured by the
comparative cycle threshold method (⌬⌬CT) and was reported as the n-fold
difference relative to the normalized expression of unstimulated samples
(37). Primers and TaqMan MGB probes for ␤-actin, IFN-␣4, and IFN-␤
have been previously described (38, 39) and were purchased from Applied
Biosystems.
Statistical analysis
Kaplan-Meier survival curves were compared using the log rank test (JMP
Software; SAS Institute) on an Apple Macintosh computer. The evaluation
of differences in cytokine levels was performed with one-way ANOVA and
Student-Newman-Keuls test. The evaluation of differences in organ CFU
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FIGURE 2. Effects of type I and II IFN signaling on
the outcome of GBS infection in adult mice. A, Survival
of WT (129Sv/Ev), IFN-␣␤R⫺/⫺, IFN-␥R⫺/⫺, and double KO (IFN-␣␤ and IFN-␥R⫺/⫺) adult mice (18 per
group) after i.p. challenge with 2 ⫻ 107 CFU of type V
GBS strain 2603. Blood (B) and kidney (C) colony
counts after a similar challenge are also shown. D, Survival of WT (C57BL/6) and IFN-␤⫺/⫺ adult mice (18
per group) after challenge with 1 ⫻ 107 CFU of type V
GBS strain 2603. Log CFU were expressed as means ⫾
SDs of 10 determinations, each conducted on a different
animal. ⴱ, p ⬍ 0.05 vs WT mice; #, p ⬍ 0.05 vs IFN␥R⫺/⫺ mice; $, p ⬍ 0.05 vs IFN-␣␤R⫺/⫺ mice.
The Journal of Immunology
levels was performed with Mann-Whitney rank order test. Data are
shown as the means ⫾ SDs of different determinations, each conducted
on a different animal. Data were assumed to be significantly different
at p ⬍ 0.05.
FIGURE 4. GBS induce type I IFN mRNA expression in cultured peritoneal macrophages. A and B, Adherent resident peritoneal cells (5 ⫻ 106/
ml) from 129SV/Ev mice were exposed to 1 ⫻ 106 CFU/ml live GBS
(strain 2603) or L. monocytogenes or to E. coli LPS (10 ng/ml). IFN-␣4 (A)
and IFN-␤ (B) mRNA expression was measured at the indicated times by
RT-PCR. C, Effect of hemolysin deficiency on GBS-induced IFN-␤ mRNA
expression. Resident peritoneal macrophages (5 ⫻ 106/ml) from C57BL/6
mice were infected with hemolysin/cytolysin-defective GBS (GBS ␤-h/c⫺)
or with the WT parental COH-1 strain (1 ⫻ 106 CFU/ml), and IFN-␤
mRNA expression was determined by RT-PCR. E. coli LPS (10 ng/ml)
was used as positive control. Data are expressed as means ⫾ SDs of five
independent observations, each conducted on a different animal. ⴱ, p ⬍
0.05 vs uninfected peritoneal macrophages by one-way ANOVA and Student-Newman-Keuls test.
Results
IFN-␣␤R-deficient neonatal mice are hypersusceptible to GBS
infection
To study the role of type I IFN in host responses against GBS, we
infected IFN-␣␤R-deficient neonatal mice and WT 129Ev/Sv controls with a low GBS dose (20 CFU of the COH-1 strain). IFN␥R⫺/⫺ and double KO IFN-␣␤ and IFN-␥R⫺/⫺ mice were also
studied for comparison, because IFN-␥ is known to play a central
role in defenses against intracellular (3, 40) as well as extracellular
(41, 42) bacteria, including GBS (43– 45). Fig. 1A shows that all
WT neonates survived the low-dose inoculum, whereas 65% of the
IFN-␣␤R⫺/⫺ and 32% of the IFN-␥R⫺/⫺ mice died. Differences in
lethality between IFN-␣␤R⫺/⫺ and IFN-␥R⫺/⫺ mice were statistically significant. Double IFN receptor KO mice were extremely
susceptible to GBS, and all died within 72 h.
To ascertain whether increased lethality was associated with a
defective ability of the host to control in vivo GBS growth, we
determined bacterial burden at 18 and 36 h after GBS challenge
(Fig. 1, B and C). No bacteria were detected in the blood and
kidney in any of the WT mice, indicating that these animals were
capable of preventing systemic spreading of GBS from the inoculation site. In contrast, 50, 70, and 100% of, respectively, the
IFN-␥R⫺/⫺, IFN-␣␤R⫺/⫺, and double KO mice were bacteremic
at 18 h after infection. Similar data were obtained after measuring
CFU in kidneys (Fig. 1C).
Next, we determined circulating cytokine levels in infected
mice. Plasma elevations in TNF-␣ and IL-6 were found in the
receptor-deficient, but not in the WT, mice in association with
bacteremia (Fig. 1, D and E). It is likely that elevated cytokine
levels merely reflected the presence of systemic infection, which
was frequent in the immunodeficient mice and absent in the controls. Therefore, these experiments could not clarify whether type
I IFN has a regulatory role on cytokine production in the course of
GBS infection.
This first set of data indicated the following: 1) IFN-␣␤R KO
animals were more susceptible to GBS-induced lethality than WT
mice, secondary to a relative inability to control systemic bacterial
spreading from a local focus; 2) this defect was marked, with lethality surpassing that seen in IFN-␥R-deficient mice; and 3) mice
lacking both type I and II IFN receptors displayed an additive
phenotype and were extremely susceptible to low-dose infection.
Effects of IFN-␣␤R deficiency in GBS-infected adult mice
Because early age is associated with a number of defects in host
defenses, it could not be excluded that the IFN-␣␤R deficiency
phenotype observed in the neonatal mouse model of GBS disease
was at least in part age related. Moreover, GBS infections are
frequent in both neonates and adults (24, 46, 47). For these reasons, we tested IFN-␣␤R-defective adult mice for susceptibility to
GBS. As in previous studies (26), much higher GBS doses (i.e.,
2 ⫻ 107 CFU of the type V strain 2603) were used in adults,
relative to the neonates, reflecting the higher resistance of the
former to infection. Under these conditions, 60, 80, and 100% of
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FIGURE 3. GBS induce type I IFN mRNA expression during infection.
The 129SV/Ev adult mice were infected i.p. with 1 ⫻ 108 CFU of type V
GBS strain 2603, and the spleens were obtained at the indicated times.
Expression of IFN-␣4 or IFN-␤ was evaluated by real-time RT-PCR after
RNA extraction (upper panel). Lower panel, Shows bacterial numbers in
the spleen. Data represent n-fold increases in mRNA expression relative
to uninfected animals. Data are expressed as means ⫾ SDs of five
independent observations, each conducted on a different animal. ⴱ, p ⬍
0.05 vs uninfected mice by one-way ANOVA and Student-NewmanKeuls test.
3129
3130
IFN-␣␤ AND BACTERIAL INFECTIONS
the IFN-␥R⫺/⫺, IFN-␣␤R⫺/⫺, and double KO mice, respectively,
succumbed to infection, whereas all WT mice survived (Fig. 2A).
As in the neonates, increased lethality was associated with systemic spreading of GBS (Fig. 2, B and C). Therefore, very similar
effects of IFN I receptor deficiency were observed in adult and
neonatal models of infection.
Role of IFN-␤ in GBS infection
The above data indicated that type I IFN is of crucial importance
in restricting GBS replication in vivo. However, these results did
not differentiate between the roles of IFN-␣ and IFN-␤. We therefore investigated the effects of deletion of the IFN-␤ gene on the
progression of GBS infection. Adult IFN-␤⫺/⫺ mice were challenged i.p. with a GBS dose (1 ⫻ 107 CFU), found in previous
studies to be sublethal for control WT C57BL/6 mice (26). Under
these conditions, IFN-␤ gene-deleted mice were significantly more
susceptible to infection in terms of lethality (Fig. 2D). Moreover,
GBS were detected in the blood and kidneys of IFN-␤⫺/⫺ mice
(with log CFU values of 3.33 ⫾ 2.79/ml and 3.08 ⫾ 2.63/kidney,
respectively, means ⫾ SD of eight determinations each conducted
on a different animal), but not in WT mice at 18 h after GBS
challenge. These data indicated that the inability of type I receptordeficient mice to control GBS infection was at least partially accounted for by defective IFN-␤ signaling.
GBS induce production of type I IFN in the spleen and in
peritoneal macrophages
Because the above data demonstrated a critical role of type I IFN
signaling in host defenses to GBS, it was of interest to determine
whether GBS stimulate type I IFN responses. To determine
whether IFN-␣ or IFN-␤ mRNA is expressed during GBS infection, spleens of WT mice (129Ev/Sv) were removed for RT-PCR
analysis at different times after i.p. challenge. In these experiments,
the animals were injected with 1 ⫻ 108 CFU, a dose resulting in
systemic infection. Under these conditions, we detected significant
increases over baseline values in the expression of both IFN-␣4
and IFN-␤ mRNA at 18 –24 h after challenge (Fig. 3, upper panel),
in association with the presence of GBS in the spleen (Fig. 3, lower
panel).
Because macrophages have a central role in innate immunity
and are abundant in the spleen, we next examined whether they
express type I IFNs in vitro in response to GBS. After adherent
peritoneal cells from 129Ev/Sv mice were exposed to live GBS,
IFN-␣4 and IFN-␤ mRNA expression was measured by RT-PCR.
LPS and L. monocytogenes, which are known to induce type I IFN
in macrophages, were included for comparison. IFN-␣4 mRNA
moderately increased within 4 h after GBS stimulation, reached the
highest levels at 6 h, and returned to normal at 12 h (Fig. 4A).
Moreover, GBS could induce even higher elevations in IFN-␤
mRNA levels (Fig. 4B). The kinetics of IFN-␣␤ induction by GBS
or Listeria was delayed relative to LPS. The degree and kinetics of
IFN-␣4 and IFN-␤ mRNA elevations after GBS stimulation were
similar in macrophages from IFN-␣␤R⫺/⫺ and WT mice (data not
shown). In additional experiments, we investigated whether increased IFN expression occurred not only at the mRNA, but also
at the protein level. To this end, IFN-␣ and IFN-␤ concentrations
were determined by ELISA in macrophage supernatants collected
at 24 h after stimulation with GBS, using the same conditions of
the experiments depicted in Fig. 4. We measured 102 ⫾ 18
pg/ml IFN-␤ in the supernatants of GBS-stimulated macrophages (means ⫾ SD of five separate experiments), whereas
cytokine levels were below the limit of detection of the assay in
unstimulated macrophages. In contrast, in both GBS-stimulated
and unstimulated macrophages, IFN-␣ was below the limit of
detection of the assay (data not shown). This was not surprising,
because only moderate IFN-␣4 mRNA elevations were previously detected by RT-PCR, which is generally considered a
more sensitive assay than ELISA (Fig. 4A). Collectively, these
findings indicated that GBS are capable of inducing type I IFN
expression both during in vivo infection and in in vitro cultured
macrophages.
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FIGURE 5. IFN-␤ signaling amplifies
macrophage responses to GBS. Upper panels, Responses in resident peritoneal macrophages from 129SV/Ev (WT), IFN-␣␤R⫺/⫺,
and IFN-␥R⫺/⫺ mice. Middle panels, Responses in resident peritoneal macrophages
from C57BL/6 (WT) and IFN-␤-defective
adult mice. Monolayers (1 ⫻ 106 cells) were
infected with the indicated CFU of GBS
strain 2603 and cultured for 24 h. Culture
supernatants were assayed for TNF-␣ (A and
D), IFN-␥ (B and E), or NO (C and F) production. The levels of these mediators were
always below the limits of detection of the
assays in supernatants from unstimulated
macrophages. Lower panels, Effect of the addition of various doses of rIFN-␤, simultaneously to GBS (1 ⫻ 106 CFU), on the production of TNF-␣ (G) or IFN-␥ (H) in
macrophages from IFN-␤-defective mice.
Data represent means ⫾ SDs of five separate
and duplicate determinations. ⴱ and #, p ⬍
0.05 relative to WT and IFN-␣␤R⫺/⫺ peritoneal macrophages, respectively, by oneway ANOVA and Student-Newman-Keuls
test. N ⫽ nondetectable.
The Journal of Immunology
3131
Proinflammatory and antibacterial responses to GBS are
severely impaired in the absence of type I IFN signaling
IFN-␤ induction by Listeria is dependent on the production of
listeriolysin, a pore-forming toxin, which mediates bacterial escape in the cytosol. Because GBS possess hemolysin, which is also
a pore-forming toxin, in additional experiments we examined
whether hemolysin had a role in IFN-␤ induction. This was not the
case, because a GBS mutant defective in hemolysin was equally
potent, relative to the parental WT strain, in its ability to induce
IFN-␤ mRNA expression in macrophages (Fig. 4C).
FIGURE 7. Type I IFN signaling amplifies macrophage responses to S. pneumoniae, E. coli, or purified
LPS. Thioglycolate-elicited peritoneal macrophages
(1 ⫻ 106 cells) from 129SV/Ev (WT) or IFN-␣␤R⫺/⫺
mice were infected with the indicated CFU of S. pneumoniae or E. coli or stimulated with E. coli LPS (10 and
100 ng/ml). After culture, supernatants were assayed for
TNF-␣ (A–C) and IFN-␥ (D–F). The levels of these mediators were always below the limits of detection of the
assays in supernatants from unstimulated macrophages.
Columns and bars represent means ⫾ SDs of five separate and duplicate determinations. ⴱ, p ⬍ 0.05 relative
to WT macrophages, respectively, by one-way ANOVA
and Student-Newman-Keuls test. ND, Nondetectable.
Type I IFN is required for host resistance against pneumococci
and E. coli
The above data showing the importance of type I IFN in GBS
infection were in sharp contrast with the detrimental role of this
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
FIGURE 6. Type I IFN signaling is required for resistance against S.
pneumoniae and E. coli. Survival of WT (129Sv/Ev) and IFN-␣␤R⫺/⫺
adult mice after i.v. (A) and intracranial (B) challenge with S. pneumoniae.
C and D, Blood colony counts in mice challenged, as described above, with
S. pneumoniae. E and F, Survival of mice challenged i.v. with two different
doses of E. coli K1. ND, Nondetectable.
Macrophages are a major source of proinflammatory cytokines,
including TNF-␣ and IFN-␥, which are known to be essential for
host defenses against GBS (26, 43– 45). Moreover, macrophages
produce NO, an important antibacterial factor, after GBS stimulation. Therefore, we tested the hypothesis that the hypersusceptibility to GBS of IFN-␣␤R-deficient mice is associated with decreased macrophage TNF-␣, IFN-␥, and/or NO production.
Resident peritoneal macrophages from IFN-␣␤R⫺/⫺ or WT
129Ev/Sv mice were stimulated with different GBS doses, and
supernatants were collected after 24 h for cytokine assays (Fig. 5,
upper panels). Because macrophage production of IFN-␥ is known
to amplify, in an autocrinous fashion, proinflammatory cytokine
responses (including the IFN-␥ response), IFN-␥R⫺/⫺ macrophages were also included for comparison. Moreover, similar experiments were conducted on macrophages from IFN-␤⫺/⫺ mice
and WT C57BL/6 controls (Fig. 5, lower panels).
A similar, marked decrease in TNF-␣ and NO production was
found in macrophage supernatants from either IFN-␣␤R- or IFN␥R-deficient macrophages after GBS stimulation, compared with
WT cells (Fig. 5, A and C). Strikingly, no or very little IFN-␥ was
detected in the supernatants of IFN-␣␤R⫺/⫺ cells, whereas robust
production was observed in WT macrophages (Fig. 5B). The defect in IFN-␥ production observed in IFN-␣␤R⫺/⫺ macrophages
was significantly more pronounced than that observed in IFN-␥Rdeficient cells. The results observed in macrophages from IFN-␤deficient mice were similar to those observed with IFN-␣␤R⫺/⫺
cells and showed significantly impaired TNF-␣, IFN-␥, and NO
responses to GBS (Fig. 5, middle panels). Moreover, the addition
of rIFN-␤ at the same time of bacterial stimulation restored the
ability of macrophages to produce TNF-␣ or IFN-␥ in response to
GBS (Fig. 5, lower panels). These data indicate that type I IFN,
and specifically IFN-␤, plays an important role in GBS-induced
production of optimal levels of TNF-␣, IFN-␥, and NO.
IFN-␣␤ AND BACTERIAL INFECTIONS
3132
Discussion
The role of IFN-␣␤ responses in resistance to bacterial pathogens
has been studied previously in vivo using two intracellular pathogens, namely M. tuberculosis and L. monocytogenes. Type I IFN
signaling had only minor effects on the outcome of experimental
lung tuberculosis (15), whereas it was detrimental in listeriosis
(16 –19). We show in this study that defective type I IFN signaling
results in impaired host resistance to different extracellular pathogens, including GBS, S. pneumoniae, and E. coli. The effect of
IFN-␣␤R deficiency was marked, with mortality surpassing that
seen in the absence of IFN-␥ signaling, a function considered crucial for host defenses against intracellular (3), as well as extracellular (41, 42, 44) bacteria. Interestingly, IFN-␣␤R⫺/⫺ and IFN␥R⫺/⫺ double KO mice showed an extremely severe, additive
phenotype and quickly succumbed to challenge with few CFU of
GBS. Collectively, our data suggest that IFN-␣␤ is a fundamental
factor for host resistance against highly frequent bacterial pathogens. Moreover, in this function, the role of IFN-␣␤ is nonredundant and complementary with that of IFN-␥.
In this study, the inability of IFN-␣␤R-deficient mice to control
GBS infection was at least partially accounted for by defective
IFN-␤ signaling, as shown by increased lethality and bacterial burden in IFN-␤ gene-deleted mice. However, a role for other members of the type I IFN family cannot be formally excluded from our
data, because GBS induced not only IFN-␤, but also IFN-␣4
mRNA both in vivo and in vitro.
The protective effects of type I IFN could be related in this study
to its ability to boost the antibacterial responses of macrophages. In
the absence of IFN-␣␤ signaling, the production of TNF-␣ and
IFN-␥ was strikingly impaired after exposure to each of the patho-
gens tested. Because both TNF-␣ and IFN-␥ are fundamental mediators of antibacterial host defenses (26, 41, 44), the increased
susceptibility to infection of IFN-␣␤R⫺/⫺ mice can be at least
partly explained by defective production of these cytokines. Moreover, the production of NO, which has direct antimicrobial effects,
was severely defective in the absence of IFN-␣␤R. It was found in
this study that IFN-␣␤ signaling was required for optimal macrophage responses to purified LPS, although such requirement was
no longer apparent using high LPS doses (Fig. 7). This dose dependency may explain the different results of two previous studies
showing reduced and unchanged TNF-␣ responses in IFN-␤-deficient mice using, respectively, LPS-stimulated macrophages (48)
and a high-dose endotoxin shock model (38).
To our knowledge, this is the first study showing in macrophages that IFN-␣␤ is required for the production of high levels of
IFN-␥. In this activity, IFN-␣␤ was significantly more potent than
IFN-␥ itself, which is known to act autocrinously on APCs in a
positive feedback loop (3). Thus, our data extend to macrophages
previous observations that IFN-␣␤ responses up-regulate IFN-␥
production in dendritic cells (49) and T cells (50 –52). However, it
cannot be excluded from our data that, in addition to macrophages,
small quantities of contaminating cells participated to IFN-␣␤dependent IFN-␥ responses. Moreover, given the role of constitutive IFN-␣␤ signals for efficient IFN-␣␤ induction by viruses (2),
further studies are required to ascertain the relative importance of
constitutive vs induced type I IFN production in boosting macrophage responses to bacteria.
We show in this study that type I IFN expression can be induced in
murine macrophages by bacteria, such as GBS, which do not have an
intracytosolic lifestyle resembling that of L. monocytogenes. The latter, by virtue of listeriolysin, can lyse endosomal membranes and
escape into the cytosol, where its presence is sensed by as yet undefined cytoplasmic receptors, leading to IFN-␤ induction. However, no
endosomal escape mechanism has been described for GBS, although
leakage of bacterial products to the cytosol cannot be discounted a
priori. Moreover, although L. monocytogenes-induced IFN-␤ responses are listeriolysin dependent, a GBS mutant defective in hemolysin, a listeriolysin homologue, was fully capable in this study of
inducing IFN-␤. Traditionally, bacterial induction of IFN-␤ has been
considered solely a function of the LPS component of Gram-negative
bacteria, based on observations that high doses (100 ␮g/ml) of heatkilled Gram-positive bacteria were unable to induce IFN-␣␤ expression in macrophages (53). We found, however, that, in addition to live
bacteria, low (0.1 ␮g/ml), but not high (ⱖ10 ␮g/ml), doses of heatkilled bacteria can also induce IFN-␣␤ expression in macrophages
(our unpublished observations). Heat-killed GBS were previously
found to induce TNF-␣ via a pathway involving MyD88, an adaptor
protein that is involved in signaling by most TLRs (54). Studies are
underway to determine whether MyD88 or TLRs are involved in
GBS-induced IFN-␣␤ expression.
In conclusion, our data indicate that type I IFN is a crucial host
defense factor against common extracellular bacterial pathogens.
A major function of this mediator was to prime macrophages for
increased proinflammatory responses. Our data support the possibility that type I IFN plays a fundamental role in host defenses
against bacterial pathogens.
Disclosures
The authors have no financial conflict of interest.
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