Commensal Bacteria Calibrate the Activation
Threshold of Innate Antiviral Immunity
Michael C. Abt,1Lisa C. Osborne,1Laurel A. Monticelli,1Travis A. Doering,1Theresa Alenghat,1Gregory F. Sonnenberg,1
Michael A. Paley,1Marcelo Antenus,2Katie L. Williams,4Jan Erikson,4E. John Wherry,1,* and David Artis1,3,*
1Department of Microbiology and Institute for Immunology
2Department of Otorhinolaryngology, Perelman School of Medicine
3Department of Pathobiology, School of Veterinary Medicine
University of Pennsylvania, Philadelphia, PA 19104, USA
4The Wistar Institute, Philadelphia, PA 19104, USA
*Correspondence: firstname.lastname@example.org (E.J.W.), email@example.com (D.A.)
Signals from commensal bacteria can influence
immune cell development and susceptibility to
infectious or inflammatory diseases. However, the
mechanisms by which commensal bacteria regulate
protective immunity after exposure to systemic
pathogens remain poorly understood. Here, we
demonstrate that antibiotic-treated (ABX) mice
immune responses and substantially delayed viral
clearance after exposure to systemic LCMV or
mucosal influenza virus. Furthermore, ABX mice ex-
hibited severe bronchiole epithelial degeneration
and increased host mortality after influenza virus
infection. Genome-wide transcriptional profiling of
macrophages isolated from ABX mice revealed
decreased expression of genes associated with anti-
viral immunity. Moreover, macrophages from ABX
mice exhibited defective responses to type I and
type II IFNs and impaired capacity to limit viral
replication. Collectively, these data indicate that
commensal-derived signals provide tonic immune
stimulation that establishes the activation threshold
of the innate immune system required for optimal
Commensal microbial communities colonize barrier surfaces
of the skin, vaginal, upper respiratory, and gastrointestinal
tracts of mammals and consist of bacteria, fungi, protozoa,
and viruses (Breitbart et al., 2003; Ley et al., 2006a; Scupham
reside in the intestine and have beneficial properties ranging
from aiding in metabolism to competing with invasive pathogens
for the environmental niche (Honda and Littman, 2012; Sonnen-
burg et al., 2006). Studies in patients have associated alterations
in bacterial communities with susceptibility to diabetes, obesity,
cancer, inflammatory bowel disease (IBD), allergy, and other
atopic disorders, highlighting the potential impact of host-
commensal interactions on multiple metabolic and chronic
inflammatory diseases (Ley et al., 2006b; Manichanh et al.,
2006; Moore and Moore, 1995; Penders et al., 2007).
(ABX), or selectively colonized mice have demonstrated that
deliberate manipulation of commensal bacterial communities
results in impaired lymphoid tissue development, dysregulated
immune cell homeostasis, and altered susceptibility to infectious
tis, 2009; Littman and Pamer, 2011; Smith et al., 2007). For
example, experimental colonization of mice with Clostridium
spp. induced CD4+regulatory T cells in the intestine and amelio-
rated intestinal inflammation in a murine model of IBD (Atarashi
et al., 2011). In contrast, colonization of the intestine with
segmented filamentous bacteria (SFB) is associated with
increased frequencies of intestinal CD4+T helper 17 cells and
exacerbated autoimmune inflammation in murine models of
arthritis, multiple sclerosis, and diabetes, demonstrating that
2010). Consistent with proinflammatory properties, signals from
commensal bacteria can act as an adjuvant, augmenting
immune responses after intestinal parasitic or bacterial infec-
tions (Benson et al., 2009; Hall et al., 2008; Ivanov et al., 2009).
Conversely, commensal bacteria can increase viral infectivity in
the gastrointestinal microenvironment (Kane et al., 2011; Kuss
et al., 2011). Thus, commensal-derived signals are capable of
limiting or exacerbating infection in the intestinal microenviron-
ment. However, the mechanisms through which commensal-
derived signals regulate innate and adaptive immunity to infec-
tion remain poorly defined.
The mammalian innate immune system has evolved diverse
strategies each tailored to detect and respond to distinct patho-
gens. Despite this apparent specialization, crosstalk between
pathways has been reported in which stimulation from one class
of pathogens influences the response to another (Barton et al.,
2007; Spencer et al., 1977). However, it is unclear whether
commensal bacteria influence innate immune pathways in the
steady-state and, if so, whether these interactions modulate
responsiveness to viral pathogens. Iwasaki and colleagues
reported impaired antiviral immunity in the lung after manipula-
tion of commensal bacteria (Ichinohe et al., 2011) that was
158 Immunity 37, 158–170, July 27, 2012 ª2012 Elsevier Inc.
associated with defective activation of the inflammasome
(Lamkanfi and Dixit, 2011). Whether depletion of commensal
bacteria selectively regulates inflammasome-dependent path-
ways or represents broader immunological crosstalk between
commensal bacteria and antiviral pathways remains to be
In this report, we examine this fundamental question and
demonstrate that manipulating commensal bacteria results in
impaired host protective immunity
(lymphocytic choriomeningitis virus [LCMV]) or mucosal (influ-
enza virus) infection, leading to dysregulated adaptive immune
responses and underlying defects in innate antiviral pathways.
Genome-wide transcriptional profiling of macrophages from
naive ABX mice revealed reduced expression of genes associ-
ated with IFN activation and antiviral immunity. Moreover,
macrophages from ABX mice exhibited impaired responsive-
ness to type I and type II IFNs and a reduced capacity to control
viral replication. Restoration of IFN responsiveness in ABX mice
re-established protective antiviral immunity after influenza virus
infection. Taken together, these data indicate that commensal
bacteria provide tonic signals that calibrate the activation
threshold and sensitivity of the innate antiviral immune system.
Defective Immunity to LCMV Infection after
ABX-Mediated Depletion of Commensal Bacteria
Signals from commensal bacteria regulate intestinal immune cell
homeostasis in multiple settings (Hill and Artis, 2010; Round
and Mazmanian, 2009); however, whether commensal-derived
signals regulate immunity to pathogens that infect sites other
than the gastrointestinal tract is unclear. To address this ques-
tion, we administered naive C57BL/6 mice oral doses of
broad-spectrum antibiotics for 2 weeks and subsequently in-
fected them with LCMV T1b, a strain of virus that causes viremia
for 1–2 weeks and that requires a robust innate and adaptive
immune response for viral clearance (Blackburn et al., 2009).
As reported in our earlier studies, exposure to antibiotics re-
sulted in a reduction in intestinal commensal bacteria and
dramatic reorganization of the bacterial community structure
(Hill et al., 2012). After infection with LCMV, conventionally
housed (CNV) mice exhibited maximal viremia at day 7 (d7)
post-infection (p.i.) and successfully controlled viremia by d23
(Figure 1A). Control of infection was associated with expansion
of LCMV-specific CD8+T cells (Figures 1B and 1C) and LCMV-
specific IgG in the serum (Figure 1D). In contrast, ABX mice
exhibited a significant delay in clearance of circulating virus
(Figure 1A, p = 0.036) and increased viral titers in the kidneys
at d31 p.i. (Figure S1A available online). Impaired viral control
was associated with reduced LCMV-specific CD8+T cell
responses and IgG antibody titers in the blood (Figures 1B–
1D). In addition, at d31 p.i., LCMV-specific H2-DbGP33
tetramer+CD8+T cells isolated from ABX mice expressed
increased levels of the inhibitory receptors PD-1, 2B4, CD160,
and LAG-3 (Figure 1E) and were less efficient producers of
multiple effector molecules (IFN-g, TNF-a, IL-2, MIP-1a, and
CD107a) (Figures 1F and 1G). Analysis of CD8+T cell responses
specific for other LCMV epitopes revealed similar impairment
of the total LCMV-specific CD8 T cell response in ABX mice
(Figures S1B and S1C). These results are consistent with
more severe T cell exhaustion in the ABX group, a characteristic
sign of impaired immunity to LCMV infection (Wherry, 2011).
signals results in defective virus-specific adaptive immune
responses and inefficient control of viral replication after
Enhanced Susceptibility and Reduced Immunity
to Influenza Virus Infection in ABX Mice
To test whether commensal bacteria might influence optimal
host defense following exposure to other viral infections, we
infected CNV or ABX mice with influenza virus (PR8-GP33)
and analyzed immunologic, virologic, and pathologic parame-
ters. Similar to the gastrointestinal tract (Figure S2A), there
was a loss of culturable aerobic and anaerobic commensal
bacteria in the upper respiratory tract of ABX mice compared
to CNV mice (Figure S2B). After exposure to influenza virus
infection, CNV mice lost ?20% of their original body weight
(Figure 2A) and had reduced lung function as measured by
blood oxygen saturation (Figure 2B). Approximately 80% of
CNV mice recovered and cleared virus from the lungs by d12
p.i. (Figures 2C and 2D). Histopathological examination of lung
sections from CNV mice at d12 p.i. (Figures 2E and 2F)
compared to uninfected mice (Figure S2C) revealed peribron-
chiolar inflammation and epithelial hyperplasia, indicating
ongoing tissue damage and repair (Figures 2E and 2F). In
contrast, ABX mice lost significantly more weight (Figure 2A,
p = 0.013), had a drastic drop in blood oxygen saturation (Fig-
ure 2B), exhibited significantly higher viral titers in the lung (Fig-
ure 2C, p = 0.016), and had increased mortality (Figure 2D, p %
0.001) after influenza virus infection. Lung sections from in-
fected ABX mice revealed more pronounced epithelial cell
necrosis (Figures 2G and 2H), increased exudate and dead cells
in the bronchiolar lumen (Figure 2I, arrows), and in the most
severe cases, complete loss of the bronchiole epithelial layer
(Figure 2J, arrows). Scoring of histological sections of the lung
confirmed increased prevalence of epithelial cells with morpho-
logic features of degeneration and necrosis in ABX mice relative
to CNV mice (Figure 2K). Consistent with this, increased cell
death was observed in the bronchiole alveolar lavage (BAL) fluid
of ABX compared to CNV mice (Figures S2D and S2E). Similar
to a previously published report (Dolowy and Muldoon, 1964),
influenza virus-infected germ-free (GF) mice also exhibited
increased weight loss (Figure S2F), impaired viral clearance
(Figure S2G), reduced virus-specific antibody titers (Figure S2H),
and more severe bronchiole epithelial degeneration compared
to CNV mice (Figures S2I and S2J). In addition, CNV or ABX
mice were infected with X31-GP33 virus, a less pathogenic
strain of influenza virus that causes minimal morbidity and
mortality in CNV mice (Bouvier and Lowen, 2010; Decman
et al., 2010). Consistent with results using the PR8 strain of
influenza virus, ABX mice exhibited considerably greater weight
loss (Figure S3A), elevated viral titers (Figure S3B), increased
lung epithelial degeneration (Figure S3C), and increased
mortality (Figure S3D). Taken together, these data indicate
that commensal-derived signals are critical in promoting
optimal immunity against multiple viral infections at sites distinct
from the gastrointestinal tract.
Commensal Bacteria Regulate Antiviral Immunity
Immunity 37, 158–170, July 27, 2012 ª2012 Elsevier Inc. 159
Diminished Generation of Influenza Virus-Specific
Adaptive Immunity in ABX Mice
Analysis of the adaptive immune response revealed that CNV
mice infected with PR8-GP33 virus generated large populations
of influenza virus-specific CD8+Tcellsin the BAL(Figure3A) and
lung parenchyma (Figures 3B and 3C) by d7 p.i. In contrast, the
total number of influenza virus-specific CD8+T cells present in
these tissueswasreduced in ABXmice (Figures 3A–3C). Consis-
tent with these data, infection with X31-GP33 also resulted in
significantly fewer tetramer+CD8+T cells in the BAL (Figure S3E,
p = 0.034), lung parenchyma (Figure S3F, p = 0.008), mediastinal
0.001) of ABX mice compared to CNV mice. Additionally, virus-
specific CD8+T cells in the lung of ABX mice were less capable
of producing multiple effector molecules simultaneously (IFN-g,
TNF-a, IL-2, MIP-1a, and CD107a) (Figure S3I). ABX mice also
exhibited lower titers of PR8-specific IgM and IgG in the serum
Expression of activation markers such as CD69, CD25, and
Granzyme-B is rapidly upregulated early after viral infection and
then downregulated as the infection is controlled (Lawrence
and Braciale, 2004; Wherry et al., 2007). Although DbGP33
tetramer+CD8+T cells from CNV mice downregulated early
activation markers between d7 to 12 p.i., expression of CD69
and CD25 remained elevated in DbGP33 tetramer+CD8+
T cells isolated from ABX mice (Figure 3E; Table S1). Elevated
expression of these molecules, along with Granzyme-B and
with a delay in the adaptive immune response to influenza virus
and sustained activation of virus-specific CD8+T cells due to
Figure 1. Systemic LCMV T1b Infection Results in Delayed Viral Clearance and an Impaired LCMV-Specific CD8+T Cell Response in
(A) Viral titer in the serum of CNV or ABX C57BL/6 mice after LCMV T1b infection (L.o.D., limit of detection).
(B and C) LCMV-specific (B) DbGP33 and (C) DbGP276 tetramer+CD8+T cells per 106peripheral blood mononuclear cells (PBMC) at d7 and d14 p.i.
(D) Serial dilution of LCMV-specific IgG antibody titers in serum of CNV or ABX mice at d23 p.i. Naive serum from CNV mice used for baseline.
(E) Expression of inhibitory receptors PD-1, 2B4, CD160, LAG-3 on DbGP33 tetramer+CD8+T cells isolated from the spleen of CNV (black line) or ABX (red line)
mice. Shaded histograms represent CD44loCD8+T cells. Numbers in italics represent mean fluorescence intensity (MFI).
(F and G) Splenocytes from d31 infected mice were incubated with GP33 peptide for 5 hr in the presence of BFA and assessed for production of IFN-g, TNF-a,
MIP-1a, CD107a, and IL-2. FACS plots gated on live, CD8a+cells.
(G) Proportion of GP33 peptide responsive CD8+T cells producing multiple effector molecules. Data representative of three independent experiments with
n = 5 mice per group. Data shown are the mean ± SEM. Serum viral titer statistics determined by two-part t test for each time point. *p < 0.05, **p < 0.01. See also
Commensal Bacteria Regulate Antiviral Immunity
160 Immunity 37, 158–170, July 27, 2012 ª2012 Elsevier Inc.
prolonged viral replication. Collectively, these data demonstrate
ties result in impaired humoral and cellular immune responses
necessary for clearing systemic or mucosal viral infections.
Impaired Innate Antiviral Immune Responses
in ABX Mice
The impaired antiviral CD8+T cell response in ABX mice after
LCMV or influenza virus infection suggested a potential defect
in the early innate immune response. To test this, we assessed
cells after PR8-GP33 virus infection. There was a comparable
influx of macrophages, inflammatory monocytes, neutrophils,
plasmacytoid dendritic cells (pDC) (Figures 4A and S4A), and
conventional dendritic cells (cDC) (Figure 4B) into the BAL, lung
or draining lymph nodes of CNV versus ABX mice. Further,
both cDC (Figure 4C) and pDC (Figure S4B) exhibited a similar
activation profile in CNV and ABX mice. However, although the
total number of macrophages was similar between CNV and
ABX mice, there was reduced expression of macrophage-asso-
ciated antiviral response genes (Ifnb, Irf7, Mx1, Oas1a, Il28b
[Ifnl], Il6, Tnfa, Ccl3 [Mip1a], and Il1b) in the lung of ABX mice
Bacterial Communities Exacerbate Lung
Pathology and Mortality to Influenza Virus
CNV or ABX C57BL/6 mice were infected i.n. with
influenza virus PR8-GP33.
(A and B) Time course of weight loss (A) and blood
oxygen (B) saturation after infection (representa-
tive exp. n = 5: y signifies mice below 70% initial
weight were sacrificed).
(C) Influenza virus genome copies in the lung at
d12 p.i. assessed by RT-PCR and displayed as
TCID50/gram of lung tissue.
(D) Survival curve after PR8-GP33 infection; CNV
n = 27, ABX n = 25.
(E–J) H&E-stained lung section of CNV (E and F) or
ABX (G–J) mice at d12 p.i. Black box and arrows
highlight (E and F) epithelial hyperplasia, (G and H)
epithelial cell necrosis, (I) cellular debris and
exudate in lumen, and (J) loss of bronchiole
epithelium (scale bar represents 50 mm in E, G, I,
and J; 20 mm in F and H).
(K) Disease score of bronchiole epithelial degen-
eration at d12 p.i. Data representative of five
independent experiments with n = 5–6 mice per
group. Survival statistics determined by log rank
test. Viral titer statistics determined by two-part
t test. *p < 0.05, **p < 0.01, and ***p < 0.001. Data
shown are mean ± SEM. See also Figure S2.
2. Alterations inCommensal
immune response. Consistent with an
following influenza virus infection, as
early as 12 hr post-LCMV infection there
was reduced expression of mRNA en-
coding Ifnb, Irf7, Mx1, Oas1a, and Stat1
in the spleen (Figure 4E) and decreased
to CNV mice (Figure 4F), indicating that diminished innate anti-
viral immune responses are a general feature in ABX mice after
either mucosal or systemic viral infection.
The immunregulatory cytokine IL-10 has been demonstrated
to influence the antiviral immune response (Brooks et al., 2006;
McKinstry et al., 2009; Sun et al., 2009) and expression of this
cytokine can be modulated by commensal bacteria (Amaral
et al., 2008; Mazmanian et al., 2008). However, prior to infection,
no differences in expression of Il10 mRNA were observed in
the spleen (Figure S4C) or lung (Figure S4D) of CNV and ABX
mice. Further, after influenza virus infection, a similar increase
in IL-10 was detected in the BAL of CNV and ABX mice (Fig-
ure S4E), indicating that impaired antiviral immune responses
in ABX mice were not associated with dysregulated IL-10
production. In contrast, proinflammatory cytokines and chemo-
kines were reduced in the BAL after influenza virus infection
(Figure S4F) or in the spleen after LCMV infection (Figure S4G)
of ABX mice early after infection. Furthermore, expression of
MHC-I and CD86 on macrophages from ABX, LCMV-infected
mice was reduced at d1 p.i. (Figure 4G). Together, these findings
indicate that ABX-mediated alterations in commensal bacterial
communities result in selective dysregulation of innate immune
responses after systemic or mucosal viral infection.
Commensal Bacteria Regulate Antiviral Immunity
Immunity 37, 158–170, July 27, 2012 ª2012 Elsevier Inc. 161
Defective Expression of Antiviral Defense Genes
in Macrophages from ABX Mice
Early defects in the antiviral response in ABX mice provoke
the hypothesis that commensal-derived signals regulate the
activation status of innate immune cells prior to viral infection.
Phenotypic characterization revealed that macrophages (Fig-
ure S5A), but not DCs (Figure S5B), isolated from the peritoneal
cavity of naive ABX mice had decreased expression of IFN-gRI,
MHC-I, CD40, and CD86 molecules that are critical during the
early response to viral infection. To interrogate the potential
mechanisms through which signals derived from commensal
bacteria regulate macrophage responses and innate antiviral
immunity, we employed genome-wide transcriptional profiling
of macrophages isolated from CNV or ABX mice prior to viral
infection. Fundamental differences in transcriptional profiles
were readily apparent between macrophages isolated from
CNV versus ABX mice (Figure S6A). In total, there were 367
gene transcripts upregulated by R1.6-fold in macrophages iso-
lated from CNV mice relative to macrophages isolated from
ABX mice (Figure 5A). Ingenuity pathways analysis identified
the interferon signaling pathway as the most significantly en-
riched pathway in macrophages isolated from CNV mice versus
ABX mice (Figure 5B, p % 0.001). In addition, gene set enrich-
ment analysis (GSEA) (Haining and Wherry, 2010; Subramanian
et al., 2005) revealed that six of the top eight most enriched
gene sets in macrophages from CNV mice were related to
interferon responses (Figures S6B–S6D). Analysis of specific
gene expression in macrophages isolated from ABX mice re-
vealed a relative downregulation of several genes that regulate
detection of virus (Ifih1 [Mda5] and Ddx58 [Rig-I]), the response
to interferon signaling (Irf7, Ifngr1, Stat1, and Stat2), or inhibi-
tion of viral replication (Mx1 and Oas1a) (Figure 5C), but not
genes associated with the inflammasome or TLR signaling
pathways (Figures S6E and S6F), two alternative innate path-
ogen recognition mechanisms. Differential expression of these
independently confirmed by real-time PCR (RT-PCR) analysis
(Figure S6G). Taken together, genome-wide transcriptional
profiling and computational analyses suggest that signals
derived from commensal bacteria calibrate the activation
response pathways in
Figure 3. ABX Mice Have a Diminished Influenza Virus-Specific Adaptive Immune Response
(A and B) Total number of influenza virus-specific DbGP33 tetramer+CD8+T cells isolated from the (A) BAL or (B) lung parenchyma at d7 p.i.
(C) DbNP366 tetramer+CD8+T cells isolated from the lung parenchyma at d7 p.i.
(D) PR8-specific IgM and IgG titers in the serum at d12 p.i.
(E) Phenotypic profile of DbGP33 tetramer+CD8+T cells isolated from the lung of CNV (solid line) or ABX (dotted line) mice at d7, d10, and d12 p.i. Gray shaded
histograms are CD44loCD8+T cells isolated from the lung. Numbers in italics represent MFI. Data representative of three independent experiments with n = 4–5
mice per group. *p < 0.05 and **p < 0.01. Data shown are mean ± SEM. See also Figure S3.
Commensal Bacteria Regulate Antiviral Immunity
162 Immunity 37, 158–170, July 27, 2012 ª2012 Elsevier Inc.
Reduced Response to IFN Stimulation or Viral Infection
in Macrophages Isolated from ABX Mice
commensal-derived signals modulate the responsiveness of
macrophages to viral infection or IFN stimulation. IFN receptor
signaling results in phosphorylation of STAT1 followed by
translocation into the nucleus, where STAT1 mediates tran-
scription of interferon-responsive genes crucial to the early
antiviral response (Schindler et al., 1992; Shuai et al., 1992).
To functionally test IFN responsiveness, we stimulated macro-
phages isolated from naive CNV or ABX mice with IFN-g or
IFN-b in vitro and measured STAT1 phosphorylation. Stimu-
lated macrophages isolated from ABX mice exhibited signifi-
cantly reduced phospho-STAT1 (pSTAT1) compared to CNV
macrophages after IFN-g (Figures 6A and 6B, p % 0.001) or
IFN-b (Figures 6C and 6D, p = 0.025) stimulation. Similar defi-
ciencies in STAT1 phosphorylation were observed after IFN-g
(Figures S7A and S7B) or IFN-b (Figures S7C and S7D) stim-
ulation of macrophages isolated from GF mice. To determine
whether defects in IFN responsiveness impact the response
to viral infection, we infected macrophages isolated from naive
CNV or ABX mice with influenza virus in vitro and examined
induction of antiviral defense genes. Expression of multiple
antiviral defense genes (Mx1, Oas1a, Ifnb, and Stat2) was
reduced in macrophages isolated from ABX mice compared
to CNV mice at 6–24 hr p.i. (Figure 6E), indicating an inherent
inability to respond to viral infection. Furthermore, macro-
phages isolated from ABX (Figure 6F, p = 0.04) or GF
(Figure S7E, p % 0.01) mice supported significantly more
viral replication upon in vitro infection with LCMV. These
biological observations are consistent with diminished expres-
sion of antiviral defense genes identified by transcriptional
Figure 4. Innate Antiviral Immune Response Is Diminished in ABX Mice after Influenza Virus or LCMV Infection
(A) Total numbers of alveolar macrophages, inflammatory monocytes, neutrophils and plasmacytoid dendritic cells isolated from BAL at d3 p.i.
(B) Total numbers of dendritic cells (DCs) isolated from the lung parenchyma and mediastinal lymph node (medLN).
(C) Expression of CD86 and CD40 on naive or d3 p.i. DCs isolated from the lung and medLN of CNV or ABX mice.
(D) Fold induction of antiviral defense gene expression in the lung at d3 post-influenza virus infection relative to lung of naive CNV mice as assessed by RT-PCR.
(E) Fold induction of antiviral defense genes in the spleen 12 hr after LCMV (T1b) infection relative to spleen of naive CNV mice as assessed by RT-PCR.
(F) IFN-b levels in the serum at 12 hr post LCMV infection as detected by ELISA.
experiments with n = 3–5 mice per group. *p < 0.05, **p < 0.01. Data shown are mean ± SEM. See also Figure S4.
Commensal Bacteria Regulate Antiviral Immunity
Immunity 37, 158–170, July 27, 2012 ª2012 Elsevier Inc. 163
profiling (Figure 5) and indicate that signals from commensal
bacteria are crucial for maintaining optimal responsiveness
of macrophages to IFN stimulation required to control viral
Impaired In Vivo Antiviral Macrophage Response in ABX
Mice after Influenza Virus or LCMV Infection
To test whether defects in the antiviral macrophage response
exist in vivo after viral infection, we infected CNV or ABX mice
with LCMV and assessed STAT1 phosphorylation in macro-
phages. As early as 6 hr after infection, macrophages from
the spleen of ABX mice exhibited reduced pSTAT1 relative to
splenic macrophages isolated from CNV mice (Figure 7A). By
12 hr p.i., peritoneal macrophages from ABX mice exhibited
significantly decreased STAT1 phosphorylation compared to
peritoneal macrophages from CNV mice (Figure 7B, p %
0.001). To determine whether alveolar macrophages also ex-
hibited a defective antiviral response after influenza virus infec-
tion, we sorted macrophages from the BAL of CNV or ABX
mice at d3 post-influenza virus infection and assessed a panel
of antiviral defense genes by RT-PCR. Airway macrophages
from ABX mice exhibited reduced induction of antiviral defense
genes including Mx1, Stat1, Irf7, Ifit3, and Ifnb (Figure 7C), indi-
cating that the macrophage response in ABX mice was qualita-
tively impaired after viral infection.
To assess whether re-establishing IFN responsiveness in
ABX mice could restore protective immunity to influenza virus
infection, we administered poly I:C to ABX mice and then
challenged them with influenza virus. ABX mice receiving PBS
had drastic weight loss (Figure 7D), loss of lung function
(Figure 7E), and high mortality after influenza virus infection
(Figure 7F). In contrast, administration of poly I:C to influenza
virus-infected ABX mice resulted in less weight loss (Figure 7D)
as well as marked improvements in lung function and survival
(Figures 7E and 7F), suggesting that recalibrating the immune
response to IFN signaling could supplant the need for tonic
commensal-derived stimulation of the innate immune system.
Taken together, these observations indicate that commensal
bacterial communities have a fundamental role in setting the
activation threshold of the innate immune system required for
optimal antiviral immune responses.
Our studies demonstrate an unexpected role for commensal
bacteria in calibrating the responsiveness of antiviral immunity.
ABX-mediated alterations of commensal bacteria compromised
innate and adaptive immune responses after systemic or respi-
ratory viral infection. Severe defects in the adaptive immune
response to LCMV and influenza virus as well as poor control
of viral replication pointed toward early defects in the initiation
of antiviral responses. Indeed, genome-wide transcriptional
profiling and functional assays uncovered a global defect in
antiviral responsiveness of macrophages isolated from ABX
mice. Upon deliberate manipulation of commensal bacteria,
expression of antiviral defense genes and interferon responsive
pathways were altered in the steady state. For rapidly replicating
viruses, such a delay in initiating antiviral pathways and acti-
vating downstream events such as humoral and cell-mediated
adaptive immune responses can have dramatic consequences
leading to failure to control infection, increased host morbidity,
and mortality. Together, these data suggest a model in which
signals from commensal bacteria calibrate the activation
threshold of innate antiviral immune responses.
Commensal bacterial communities modulate immune cell
homeostasis and disease by providing either immunoregulatory
Figure 5. Naive Macrophages from ABX Mice Have an Attenuated Antiviral Defense Gene Profile
RNA was extracted from sort-purified peritoneal macrophages isolated from naive CNV or ABX mice. Extracted RNA was hybridized to an Affymetrix GeneChip
microarray to assess gene expression.
(A) Frequency and total number of elevated genes in CNV macrophages compared to macrophages isolated from ABX mice.
(B) Highly enriched biological pathways and functions found within the subset of elevated genes from CNV macrophages as assessed by Ingenunity pathways
analysis. Red bars indicate the percent of genes in a pathway upregulated in macrophages isolated from CNV mice. Yellow line indicates p value calculated by
Fisher’s exact test.
(C) Heat map of key antiviral defense genes in macrophages isolated from CNV or ABX mice. Red, high expression; blue, low expression. See also Figures S5
Commensal Bacteria Regulate Antiviral Immunity
164 Immunity 37, 158–170, July 27, 2012 ª2012 Elsevier Inc.
or proinflammatory signals. For example, polysaccharide A,
isolated from Bacteroides fragilis, can reduce the severity of
intestinal inflammation in two models of IBD (Mazmanian et al.,
2008). Conversely, commensal bacteria can also boost immune
responses against mucosal infections (Benson et al., 2009;
Hall et al., 2008; Ichinohe et al., 2011; Ivanov et al., 2009). These
studies provoke the hypothesis that commensal-derived signals
might influence the systemic immune response to infection. The
present study demonstrates that commensal bacteria influence
the activation threshold of broadly used innate antiviral response
pathways such as the IFN signaling pathway. Induction of a
type I IFN response is fundamental and critical for defense
against the majority of viruses (Sen, 2001). Macrophages iso-
lated from ABX mice, however, displayed major defects in
expression of key interferon-stimulated response genes even
prior to viral exposure compared to macrophages from CNV
mice. Reduction in steady-state transcription of antiviral path-
ways was associated with impaired responsiveness to type I
and type II IFNs or virus.
Iwasaki and colleagues observed that commensal bacteria
can influence inflammasome activity, an innate signaling
pathway involved in responses to bacteria, cytosolic oligomers,
Figure 6. Macrophages from Naive ABX Mice Have a Diminished Ability to Respond to IFN Stimulation and Viral Infection In Vitro
(A–D) Peritoneal Macrophages isolated from CNV or ABX mice were stimulated with IFN-g or IFN-b in vitro. Histograms of STAT1 phosphorylation in macro-
or (D) IFN-b stimulation.
(Eand F) Peritoneal macrophages sorted from naive CNV or ABX mice were infected invitro with(E) influenza virus (X31-GP33, MOI of 5)or (F) LCMV (cl-13strain,
MOI of 0.2).
(E) Induction of antiviral defense genes in macrophages at 6 and 24 hr p.i. as assessed by RT-PCR.
(F) LCMVviral titers in supernatant at 24–96 hrp.i. Data representative of two or more independent experiments with n = 3–5 mice per group. *p< 0.05, **p < 0.01,
and ***p < 0.001. Data shown are mean ± SEM. See also Figure S7.
Commensal Bacteria Regulate Antiviral Immunity
Immunity 37, 158–170, July 27, 2012 ª2012 Elsevier Inc. 165
Figure 7. In Vivo Antiviral Macrophage Response Is Impaired in ABX Mice after LCMV or Influenza Virus Infection
(A and B)CNV or ABX mice were inoculated with LCMV (T1b) or PBS i.v. and (A) splenocytes at 6hr p.i.or (B)PECs at 12hrp.i. were immediately fixed to preserve
the in vivo STAT1 phosphorylation status of macrophages.
(C) CNV or ABX mice were infected with influenza virus (PR8-GP33). At d3 p.i., alveolar macrophages were sorted from the BAL and in vivo induction of antiviral
defense genes was assessed by RT-PCR. Gene expression displayed as fold induction over naive alveolar macrophages from CNV mice. Data representative of
two independent experiments with n = 3–5 mice per group.
(D–F)CNVorABXmicewereinfectedwithinfluenzavirus(PR8-GP33). Micereceived 30mgofpolyI:C(ABX+pICgroup)orPBS(CNV&ABXgroup)i.n.atd?1and
100 mg of poly I:C or PBS i.p. at d3. Weight loss (D) and blood oxygen (E) saturation after infection (representative exp. n = 4–6: y signifies mice below 70% initial
weight were sacrificed). Weight loss statistics determined by two-way ANOVA.
(F) Survival curveafter influenza virus infection. Survivalcurve is acombination of two independent experiments.CNV n =10, ABX n =8, ABX+pIC n = 12. Survival
statistics determined by log rank test. *p < 0.05, **p < 0.01, and ***p < 0.001. Data shown are mean ± SEM.
Commensal Bacteria Regulate Antiviral Immunity
166 Immunity 37, 158–170, July 27, 2012 ª2012 Elsevier Inc.
and a subset of viruses (Ichinohe et al., 2011; Lamkanfi and Dixit,
2011). In addition, two recent reports identified a fundamental
interaction between intestinal commensal bacteria and enteric
viruses in which virus can utilize bacterial products to enhance
infectivity (Kane et al., 2011; Kuss et al., 2011). These reports
highlight the dynamic interrelationship between viral pathogens,
commensal bacteria, and the immune system. Our results reveal
a previously unrecognized interplay between commensal
bacteria and antiviral interferon signaling pathways in which
low-level tonic signaling by commensal bacteria regulates the
steady-state readiness of hard-wired antiviral pathways in
Tonic signaling has been proposed as a mechanism to main-
tain optimal responsiveness of signaling pathways in other
immunologic settings (Macia et al., 2009). For example, naive
T cells use tonic signals from low-affinity interactions with self-
MHC to regulate homeostasis and optimal dynamic responsive-
ness upon engagement of cognate antigen (Takeda et al., 1996;
Tanchot et al., 1997). In this current study, tonic signaling was
of antiviral pathways in macrophages. Although the potential
impact of antibiotic treatment on the host virome is largely
unexplored, the most direct interpretation of our data is that
commensal bacteria calibrate the threshold of innate immune
activation to viral infections and suggest steady-state innate
immune crosstalk. Such crosstalk can occur in other settings.
For example, latent viral infections can render mice less suscep-
tible to bacterial challenge, an effect attributed to basal macro-
phage activation (Barton et al., 2007). Conversely, the bacterial
species, Wolbachia, confers protection against viral infections
in Drosophila (Teixeira et al., 2008). In addition to antibacterial
defense genes, bacterial-derived LPS-TLR4 signaling can upre-
gulate transcription of antiviral genes (Amit et al., 2009; Doyle
et al., 2002). In the case of LPS-TLR4 signaling, antiviral gene
expression is initially induced, but rapidly limited by the poly-
comb repressor Cbx4 (Amit et al., 2009). This latter observation
suggests a potential explanation for the commensal-antiviral
immune fitness axis at the transcriptional level. Induction of
transcription followed by repression might maintain key antiviral
genes in a state of poised transcriptional regulation, rather than
a repressed or inactive state. Transcriptional poising, or the
presence of both activating and repressive chromatin, enables
more efficient transcriptional induction upon exposure to a true
inducer of the gene of interest (Cuddapah et al., 2010). This state
of transcriptional equilibrium provided by tonic commensal
stimulation may enable rapid induction of antiviral defense
genes upon infection. Examples of this type of regulation exist
in other biological systems such as the yeast Hog1-MAPK
pathway (Macia et al., 2009). Our results suggest that com-
mensal bacteria provide such a signal to maintain antiviral
innate immune pathways in a state of optimal readiness, allow-
ing dynamic and robust responses upon challenge by viral
It was remarkable that macrophages isolated from ABX mice
prior to viral infection also displayed less detectable in vivo
pSTAT1 compared to macrophages from naive CNV controls.
Commensal-derived signals may induce tonic, low-level STAT1
activation in the steady state, which could be a key contributing
factor to basal induction of antiviral defense genes prior to
infection. The mechanisms through which commensal-derived
signals stimulate immune cells in the periphery are poorly
understood. One possibility is that peripheral immune cells are
directly exposed to bacterial microbes or their products. Small
numbers of live commensal bacteria can be found in the Peyer’s
and there is evidence that absorbed commensal products
circulate throughout the host (Clarke et al., 2010; Macpherson
and Uhr, 2004). Thus, direct interaction between peripheral
immune cells and bacterial products is plausible. Alternatively,
commensal bacteria may act indirectly on peripheral immune
cells via responses evoked from epithelial or other mucosal-
associated stromal cells (Artis, 2008). Defining the potential
pathways involved in microbial sensing by the peripheral
immune system will be crucial for understanding how microbial
crosstalk influences immune cell homeostasis and host pro-
Modulating commensal bacterial communities has thera-
peutic potential. For example, probiotic treatments can amelio-
rate intestinal inflammatory diseases (Sartor, 2004) and success
of bacteriotherapy in cases of viral gastroenteritis demonstrates
the potential use of probiotics as a treatment strategy to combat
viral infection (Fang et al., 2009; Szajewska and Mrukowicz,
2005). Further, prophylactic probiotic administration can limit
the duration and severity of respiratory viral infections in human
subjects, suggesting that the beneficial effects of probiotics on
antiviral immunity are not limited to the gastrointestinal tract
(de Vrese et al., 2006). Despite many recent advances in defining
the diverse and dynamic microbiome in humans and animal
models of human disease, it is unclear which bacterial species
or microbial products are associated with the beneficial antiviral
effects of commensal bacteria observed in this study. It will
be important to define the commensal bacterial species and
signals that elicit these host protective effects. Such studies
could lead to new approaches for therapeutically administering
commensal bacteria or commensal-derived products and
selectively manipulating host protective immunity.
Mice and Viruses
C57BL/6 mice (4 to 6 weeks old) were purchased from the National Cancer
Institute (Frederick, MD). Mice were maintained in specific pathogen-free
facilities at the University of Pennsylvania. GF C57BL/6 and Swiss Webster
mice were maintained in plastic isolator units and fed autoclaved chow and
water. The University of Pennsylvania Institutional Animal Care and Use
Committee (IACUC) approved all protocols, and all experiments were per-
formed in accordance with the guidelines of the University of Pennsylvania
IACUC. Mice were inoculated intravenously (i.v.) with LCMV T1b (2 3 106
PFU), or intranasally (i.n.) with recombinant influenza viruses expressing the
LCMV GP33 epitope (PR8-GP33: 368 TCID50, H3N2 X31-GP33: 1 3 105
TCID50) as described (Decman et al., 2010). LCMV viral titers were determined
by plaque assay on Vero cell monolayers (Ahmed et al., 1984).
Oral Antibiotic Treatment
Mice were provided autoclaved drinking water supplemented with ampicillin
(0.5 mg/mL, Sigma), gentamicin (0.5 mg/mL, Gemini Bio-Products), metroni-
dazole (0.5 mg/mL Sigma), neomycin (0.5 mg/mL, Med-Pharmex), vancomy-
cin (0.25 mg/mL, Novaplus), and sucralose (4mg/mL, Splenda, McNeil
Nutritionals, LLC). Splenda was added to make the antibiotic cocktail more
palatable. Antibiotic treatment was started 2–4 weeks prior to infection and
continued for the duration of the experiment.
Commensal Bacteria Regulate Antiviral Immunity
Immunity 37, 158–170, July 27, 2012 ª2012 Elsevier Inc. 167
Anesthetized mice had neck and throat hair removed with a chemical depila-
tory agent (Nair, Church & Dwight Co.) 1 day prior to infection. MouseOx Tm
Pulse-Oximeter neck sensors (Starr Life Sciences, Oakmount, PA) were
placed on exposed skin and blood oxygen saturation was recorded with
Starr MouseOx software v. 5.1. Pulse oximetry readings were allowed to
stabilize and 15 seconds of measurements were averaged.
Isolation of Cells from the Spleen, Blood, Lymph Node, Peritoneal
Cavity, Lung Tissue and Airway
Lymphocytes were isolated from the spleen, lymph node, blood, lungs, or
BAL as described (Decman et al., 2010). Peritoneal exudates cells (PECs)
were obtained by injecting and recovering PBS from the peritoneal cavity.
Flow Cytometry, Tetramer, and Intracellular Staining
Single-cell suspensions were stained for surface antigens with antibodies
and tetramers or for intracellular cytokines (ICS) after peptide stimulation as
described (Decman et al., 2010). Fluorescently conjugated antibodies used
include those specific to CD3ε, CD4, CD8a, CD11c, CD19, CD25, CD43,
CD45, CD69, CD119, CD160, F4/80, Ly6c, MHC-I (H-2Kb), MHC-II (I-A I-E)
(eBioscience), CD5, CD44, CD80, CD86, Ly6g (clone 1A8), ICOS, LAG-3,
PD-1 (clone RMP1-30), (Biolegend), CD40 (BD Biosciences), CXCR3 (R&D
Systems), 2B4 (BD PharMingen), CD11b, IL-2 (eBioscience), IFN-g (BD
PharMingen), TNF-a (Biolegend), MIP-1a (R&D Systems), and granzyme B
(Invitrogen). MHC classIpeptide tetramersweremadeand usedfor identifying
virus-specific CD8+T cells (Altman et al., 1996). Alveolar macrophages were
identified as non-T, non-B, non-NK cells (NTNBNNK), and CD11c+, F4/80+.
Inflammatory monocytes were identified as NTNBNNK, CD11c?, F4/80?,
Ly6g?, Ly6c+, and CD11b+. Neutrophils were identified as NTNBNNK,
CD11c?, F4/80?, Ly6g+, and CD11b+. DCs were identified as NTNBNNK,
F4/80?, CD11c+, and MHC-IIhi. Plasmacytoid DCs were identified as
phil, CD11cint, and PDCA-1+. Peritoneal and splenic macrophages were
identified as NTNBNNK F4/80+, CD11b+Samples were collected with a LSR
II flow cytometer (Becton Dickinson). All flow cytometry data were analyzed
by FlowJo v 8.8 (Treestar). Pie charts were created with the Pestle and SPICE
programs (Mario Roederer; Vaccine Research Center, NIAID, NIH).
RNA Isolation, cDNA Preparation, and RT-PCR
RNA was isolated from cells with an RNeasy mini-kit (QIAGEN) and from lung
or spleen tissue with mechanical homogenization and TRIzol isolation (Invitro-
gen) in accordance with the manufacturer’s instructions. PA influenza-specific
primers and probes were used for determining influenza virus genome copies
on the basis of astandard curve then converted to TCID50/ gram of lung tissue.
Genes of interest were normalized to b-actin (or Hprt for whole spleen tissue)
and displayed as fold difference relative to uninfected CNV control mice.
Histological Sections and Pathology Scoring
Lungs were inflated with 4% paraformaldehyde (PFA) and embedded in
paraffin. Five micrometer sections were cut and stained with hematoxylin
and eosin. Blind scoring of H&E-stained lung tissue sections by a board-certi-
fied veterinary pathologist reflect degree of luminal exudates (0–5) and degree
of bronchiole epithelial degeneration and necrosis (0–5), for a maximum score
Cell Sorting and Microarray Data Analysis
Peritoneal macrophages from naive CNV or ABX mice were sorted directly
into TRIzol (Invitrogen) on a BD Aria (Beckson Dickson). Test sorts were
R95% pure. For microarray analysis, RNA was extracted from three sorted
biological replicates of peritoneal macrophages from naive CNV or ABX
mice. cDNA was amplified with NuGen WT Ovation Pico kit and hybridized
to Affymetrix GeneChip Mouse Gene 1.0 ST microarrays at the University of
Pennsylvania’s Microarray facility. Expression levels were summarized with
the Robust Multichip Averaging (RMA) algorithm (Irizarry et al., 2003). The
ClassNeighbors module of GenePattern (Broad Institute, Cambridge MA)
was used for identifying differentially expressed genes. Gene transcripts
with greater than 1.6-fold difference in expression were analyzed with Inge-
nuity pathway analysis software (Ingenuity Systems, www.ingenuity.com).
A right-tailed Fisher’s exact test was used for calculating a p value. GSEA
was performed as described (Subramanian et al., 2005).
In Vitro and In Vivo Phosflow STAT1 Staining of Macrophages
Adherent macrophages from PECs were stimulated with recombinant IFN-g
(R&D Systems) or IFN-b (PBL Interferonsource). After stimulation, media was
removed and replaced with 0.05% trypsin and incubated at 37?C for 2 min.
Cells were then fixed with 1.6% PFA for 10 min, permeabilized with ice
cold 90% methanol, and stained for surface markers and pSTAT1 with PE-
conjugated anti-STAT1 (pY701) antibody (BD biosciences). LCMV-infected
CNV or ABX mice were sacrificed at 6 or 12 hr p.i. We directly fixed cells in
1.6% PFA to preserve phosphorylation status and resuspended them in
90% methanol at 4?C for 30 min as described in Krutzik et al. (2005).
In Vitro Viral Infection of Peritoneal Macrophages
Sort purified macrophages isolated from naive CNV, ABX, or GF mice were
infected with either LCMV clone 13 strain (MOI of 0.2) or influenza virus
X31-GP33 strain (MOI of 5) for 1 hr. At indicated time points, supernatants
were collected so that viral titers could be assessed by plaque assay or
macrophages were directly lysed with RLT lysis buffer (QIAGEN) and RNA
was isolated as described above.
Poly I:C Administration in Influenza Virus-Infected Mice
ABX mice were administered 30 mg of polyinosinic-polycytidylic acid (poly I:C -
Sigma) i.n. at d ?1 and 100 mg i.p. at d3 post influenza virus infection (PR8-
GP33). Control CNV and ABX mice received 30 ml of PBS i.n. at d ?1 and
100 ml i.p. at d3.
Results represent means ± SEM. Statistical significance was determined by
the unpaired, two tailed Student’s t test for individual time points, two-way
ANOVA test for time course experiments, log rank test for survival curve, or
two-part t test for comparison of groups that contained samples that were
below the limit of assay detection. Statistical analyses were performed with
Prism GraphPad software v4.0 (*p < 0.05; **p < 0.01; ***p < 0.001).
Supplemental Information includes seven figures, one table, Supplemental
Experimental Procedures and can be found with this article online at
We acknowledge members of the Artis and Wherry laboratory for helpful
discussions and critical reading of the manuscript and D. Kobuley for care of
the germ-free facility. This research is supported by the US National Institutes
of Health (grants AI061570, AI087990, AI074878, AI095608, AI091759, and
AI095466 to D.A.; grants AI071309, AI078897, AI095608, AI083022,
AI077098, and HHSN266200500030C to E.J.W.; T32-AI05528 to M.C.A.;
T32-AI007532 to G.F.S and L.A.M.; T32-RR007063 and K08-DK093784 to
T.A.; T32-AI007324 to M.A.P.), Irvington Institute Postdoctoral Fellowship of
the Cancer Research Institute (L.C.O.), the Burroughs Wellcome Fund (D.A.),
the National Institute of Diabetes and Digestive and Kidney Disease, Center
for the Molecular Studies in Digestive and Liver Disease, and the Molecular
Pathology and Imaging Core (DK50306). We thank N. Cohen for assistance
with tracheal isolation and the Abramson Cancer Center Flow Cytometry
and CellSorting Resource Laboratory (partially supportedby NCIComprehen-
sive Cancer Center Support grant #2-P30 CA016520), the Wistar flow cytom-
etry core, the UPenn Vet School Pathology Service, UPenn microarray facility,
and UPenn human immunology core for invaluable technical assistance and
Received: September 15, 2011
Revised: February 22, 2012
Accepted: April 17, 2012
Published online: June 14, 2012
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