Am J Physiol Lung Cell Mol Physiol 310: L465–L475, 2016.
First published November 20, 2015; doi:10.1152/ajplung.00224.2015.
CALL FOR PAPERS
Translational Research in Acute Lung Injury and Pulmonary
Fibrosis
MicroRNA-155 regulates host immune response to postviral bacterial
pneumonia via IL-23/IL-17 pathway
Amy Podsiad,1 Theodore J. Standiford,1 Megan N. Ballinger,2 Richard Eakin,1 Pauline Park,1
Steven L. Kunkel,3 X Bethany B. Moore,1 and Urvashi Bhan1
1
Submitted 6 July 2015; accepted in final form 13 November 2015
Podsiad A, Standiford TJ, Ballinger MN, Eakin R, Park P,
Kunkel SL, Moore BB, Bhan U. MicroRNA-155 regulates host immune response to postviral bacterial pneumonia via IL-23/IL-17 pathway. Am J Physiol Lung Cell Mol Physiol 310: L465–L475, 2016. First
published November 20, 2015; doi:10.1152/ajplung.00224.2015.—
Postinfluenza bacterial pneumonia is associated with significant mortality and morbidity. MicroRNAs (miRNAs) are small, noncoding
RNAs that regulate gene expression posttranscriptionally. miR-155
has recently emerged as a crucial regulator of innate immunity and
inflammatory responses and is induced in macrophages during infection. We hypothesized upregulation of miR-155 inhibits IL-17 and
increases susceptibility to secondary bacterial pneumonia. Mice were
challenged with 100 plaque-forming units H1N1 intranasally and
were infected with 107 colony-forming units of MRSA intratracheally
at day 5 postviral challenge. Lungs were harvested 24 h later, and
expression of miR-155, IL-17, and IL-23 was measured by real-time
RT-PCR. Induction of miR-155 was 3.6-fold higher in dual-infected
lungs compared with single infection. miR-155⫺/⫺ mice were protected with significantly lower (4-fold) bacterial burden and no differences in viral load, associated with robust induction of IL-23 and
IL-17 (2.2- and 4.8-fold, respectively) postsequential challenge with
virus and bacteria, compared with WT mice. Treatment with miR-155
antagomir improved lung bacterial clearance by 4.2-fold compared
with control antagomir postsequential infection with virus and bacteria. Moreover, lung macrophages collected from patients with postviral bacterial pneumonia also had upregulation of miR-155 expression
compared with healthy controls, consistent with observations in our
murine model. This is the first demonstration that cellular miRNAs
regulate postinfluenza immune response to subsequent bacterial challenge by suppressing the IL-17 pathway in the lung. Our findings suggest
that antagonizing certain microRNA might serve as a potential therapeutic strategy against secondary bacterial infection.
miR-155; IL-17; postviral bacterial pneumonia
the most prevalent pathogens,
causing respiratory illness every winter (61). Nevertheless, the
influenza virus still accounts for 250,000 to 500,000 deaths
each year and this number increased due to emergence of the
2009 H1N1 pandemic influenza strain (42, 46, 61).
THE INFLUENZA A VIRUS IS ONE OF
Address for reprint requests and other correspondence: U. Bhan, Univ. of
Michigan Medical Center, Division of Pulmonary and Critical Care Medicine,
109 Zina Pitcher Place, 4065 BSRB, Ann Arbor, MI 48109-2200 (e-mail:
[email protected]).
http://www.ajplung.org
Although the influenza virus itself can lead to severe pneumonia, mortality is most often caused by secondary complications of the infection. A recent report on the 2009 H1N1
influenza strain indicates that nearly 30% of fatal H1N1 cases
between May 2009 and August 2009 in the United States were
associated with a secondary bacterial infection (6, 31, 48, 66).
In the United States, many of these infections have been due to
the community-acquired methicillin-resistant Staphylococcus
aureus (CA-MRSA) strain USA300 (16, 20, 24, 30). This
association of necrotizing bacterial pneumonia with antecedent
influenza virus infection is well recognized. Studies have noted
the importance of bacterial infections due to Streptococcus
pneumoniae, Haemophilus influenzae, and S. aureus in the
fatal cases associated with the 1918 influenza pandemic as well
(5, 5a, 39). The increased influenza-associated mortality from
coinfections with S. aureus among the pediatric age group
resulted in a 2008 health advisory from the Centers for Disease
Control and Prevention (CDCHAN-00268-2008-01-30ADV-N) (14).
Th17 cells have been described as producing high levels of
the proinflammatory cytokines IL-17 and IL-22 (2, 13, 29, 68).
IL-23 has been implicated in Th17 pathway regulation, proliferation, and cytokine production. STAT3 activation, driven by
IL-6 and IL-23, is required for terminal Th17 differentiation
(34, 68). In addition to CD4⫹ T cells, many innate immune
cells respond to IL-23 and are also important in both resistance
to infection and in mediating autoimmune pathology. Patients
with hyper-IgE syndrome (Job’s syndrome) were shown to
have STAT3 mutations (38). Consequently, these patients fail
to develop Th17 cells or produce IL-17A, resulting in S. aureus
infection of the skin and lung (36). These patients appear to
have enhanced susceptibility to S. aureus due to a requirement
for IL-17 signaling in the epithelium (37), suggesting a specific
role for Th17 immunity in host defense against this pathogen.
Moreover, it has recently been shown that mice challenged
with influenza A PR/8/34 H1N1 and subsequently with S.
aureus had substantially decreased IL-17 and IL-23 production after S. aureus infection. Overexpression of IL-23 in
influenza A, S. aureus-coinfected mice rescued the induction of IL-17 and markedly improved bacterial clearance.
These data confirm a role for IL-17 and IL-23 in the
clearance of infection in the lung (27).
1040-0605/16 Copyright © 2016 the American Physiological Society
L465
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Division of Pulmonary and Critical Care Medicine, Department of Medicine, University of Michigan Medical Center, Ann
Arbor, Michigan; 2Division of Pulmonary and Critical Care Medicine, Department of Medicine, Ohio State University,
Columbus, Ohio; and 3Department of Pathology, University of Michigan Medical Center, Ann Arbor, Michigan
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miR-155 IMPAIRS POSTVIRAL BACTERIAL IMMUNE RESPONSE
METHODS
Mice. Six- to eight-week-old wild-type (WT) C57BL/6 mice were
purchased from Taconic. miR-155⫺/⫺ and C57BL/6 control mice
were purchased from The Jackson Laboratory. Mice were maintained
under pathogen-free conditions. All of the studies were performed on
age- and sex-matched mice. All of the animal studies were conducted
with approval from the University of Michigan Committee on Use and
Care of Animals.
Influenza A PR/8/34 H1N1 infection. A mouse-adapted influenza A
virus strain (strain A/PR8/34: H1N1 isotype; American Type Culture
Collection) was inoculated into mice as described. Mice were infected
with 100 plaque-forming units (PFU) of influenza A PR/8/34 H1N1
(in 40 ␮l sterile PBS) from a frozen stock or were mock infected with
PBS intranasally.
S. aureus infection. S. aureus (American Type Culture Collection
49775) producing ␥-hemolysin and Panton-Valentine leukocidin was
purchased from the American Type Culture Collection. S. aureus was
cultured as detailed by American Type Culture Collection instructions
in casein hydrolysate yeast extract containing modified medium,
overnight for 18 h to stationary growth phase. Five days post-H1N1
challenge mice received 107 colony-forming units (CFU) of S. aureus
(in 50 ␮l sterile PBS) or control PBS by intratracheal instillation.
After an additional 24 h, lungs were harvested. Viral burden was
determined by quantitative real-time RT-PCR on lung RNA for viral
matrix protein as described previously (7).
mRNA extraction and real-time (TaqMan) quantitative PCR. Total
RNA from cells was isolated per the manufacturer’s protocol for the
RNAeasy Mini Kit (Qiagen, Valencia, CA). RNA amounts were
determined by spectrometric analysis at 260 nm. All primers were
designed using Primer Express software (Applied Biosystems, Foster
City, CA). Levels of mRNA were determined by real-time quantitative RT-PCR analysis using an ABI PRISM 7000 Sequence Detection
System (ABI/Perkin Elmer, Foster City, CA).
Necropsy. Mice were euthanized at various intervals after H1N1
and/or MRSA challenge by inhalation of carbon dioxide and exsanguinated and the lungs were removed.
Lung macrophage isolation. Lung macrophages (consisting of both
alveolar and interstitial macrophages) were isolated from dispersed
lung digest cells by adherence purification as previously described
(10). This population comprises both resident and exudate/recruited
macrophages, and cells are autoflourescent, CD11c⫹, F4/80⫹ (alveolar macrophages), and CD11b⫹ CD11clow (exudate macrophages) on
flow cytometry.
Antibody neutralization. IFN␥- and IL-17-neutralizing antibodies
were kindly provided by Dr. Steven Kunkel, and the protocol has been
described previously. (21). We treated the mice with 100 ␮g of
anti-IFN␥ antibody and 200 ␮g of IL-17 antibody intraperitoneally for
neutralization of IFN␥ and IL-17, respectively. The control group
received anti-mouse IgG antibody.
IL-23 reconstitution. Murine recombinant IL-23 was purchased
from R&D Systems (Minneapolis, MN). Mice were anesthetized, and
2 ␮g rmIL-23 were administered intratracheally 4 h before challenge
with MRSA.
Whole lung homogenization for CFU determination. At designated
time points, the mice were euthanized by CO2 inhalation. Before
lung removal, the pulmonary vasculature was perfused by infusing
1 ml of PBS containing 5 mM EDTA into the right ventricle.
Whole lungs were removed, with care taken to dissect away lymph
nodes. The lungs were then homogenized in 1 ml of PBS with
protease inhibitor (Boehringer Mannheim, Indianapolis, IN). Homogenates were then serially diluted 1:5 in PBS and plated on
blood agar to determine lung CFU.
Bronchoalveolar lavage. Bronchoalveolar lavage (BAL) was performed for collection of BAL fluid (BALF) as previously described
(10). Briefly, the trachea was exposed and intubated using a 1.7-mmouter-diameter polyethylene catheter. BAL was performed by instilling PBS containing 5 mM EDTA in 1-ml aliquots. A total of 3 ml PBS
was instilled per mouse, with 90% of lavage fluid retrieved.
Human macrophage isolation. BALF was collected from patients
admitted to the ICU for respiratory failure/aacute respiratory distress
syndrome (ARDS) on mechanical ventilation and who had undergone
BAL for clinical purposes; Institutional guidelines were followed and
patient samples were de-identified. Since the project involved only
biological specimens that could not be linked to a specific individual
directly or indirectly, in accordance with Office of Human Research
Protection guidance on this subject, the University of Michigan
Institutional Review Board (IRB) deemed it IRB exempt. We also
had IRB approval from the University of Michigan to collect
samples from healthy volunteers. Patient characteristics are described in Table 1. BALF was filtered and centrifuged to form cell
pellets. Patients included in our analysis were enrolled between
October 2013 and February of 2014, were ⱖ18 yr of age, met ARDS
criteria as defined by the Berlin definition (1), required endotracheal
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MicroRNAs are small RNAs that posttranscriptionally regulate eukaryotic gene expression. In addition to their involvement in a wide range of physiological and pathological processes, including viral infections, microRNAs are increasingly
implicated in the eukaryotic response to bacterial pathogens.
Recent studies have characterized changes in host microRNA
expression following infection with exclusively extracellular
(Helicobacter pylori) or intracellular (Salmonella enterica)
Gram-negative bacteria, as well as in the response to Grampositive (Listeria monocytogenes) and other pathogens (Mycobacterium and Francisella species). A role for miRNAs in the
innate immune response was demonstrated when microRNAs
such as miR-146a, miR-155, and miR-21 were shown to be
induced in response to Toll-like receptor 4 (TLR4) signaling in
monocytes (43, 45, 51, 56). miR-155 is induced by LPS, as
well as other TLR ligands and proinflammatory cytokines (45).
miR-155 has been found to be upregulated in several activated
immune cells, including T lymphocytes, B lymphocytes, macrophages, and dendritic cells (DCs). This microRNA also is
upregulated by a broad range of inflammatory mediators [such
as tumor necrosis factor (TNF)-␣, interferons, and polyriboinosinic:polyribocytidylic acid] during innate immune responses (57). The expression of miRNA-155 is upregulated
upon lymphocyte activation (19), and it has been shown to
control cell proliferation and differentiation (44, 65). For instance, miRNA-155 regulates B-cell proliferation, malignancy,
and antibody production, at least in part through inhibition of
activation-induced cytidine deaminase and PU.1 expression
(47, 60, 67). miR-155 can also negatively regulate the differentiation and function of IL-17-producing helper T cells (26,
32, 43). However, the role of miR-155 in innate immune
response to postviral bacterial pneumonia has not been studied.
In this study, we evaluated the mechanisms of impaired lung
antibacterial responses postinfluenza infection. Mice sequentially infected with influenza and MRSA have significant
upregulation of IFN␥, which leads to increased expression of
miR-155 in whole lung, and this is associated with blunted
IL-23 and IL-17 expression. miR-155 ⫺/⫺ mice demonstrate
improved bacterial clearance secondary to a more robust IL-23
and IL-17 expression in the lung. Treatment with IL-17neutralizing antibody largely abolishes the protected phenotype
seen in miR-155⫺/⫺ mice. Macrophages isolated from intubated patients in the intensive care unit (ICU) with postinfluenza bacterial pneumonia also had significantly higher expression of miR-155 compared with healthy controls.
L467
miR-155 IMPAIRS POSTVIRAL BACTERIAL IMMUNE RESPONSE
Men
Age
PaO2/FIO2 ratio
Bacterial isolates no. of patients
Staphylococcus
aureus
Streptococcus
pneumoniae
77%
55 ⫾ 12
118 ⫾ 11
9 (75%)
67%
49 ⫾ 11
109 ⫾ 28
3 (25%)
RESULTS
Mice have higher bacterial burden and poor survival after
influenza infection. In patients, the peak incidence of secondary
bacterial pneumonia occurs between 4 –10 days after the initial
influenza infection (49). In animal models, influenza infections
lead to impaired ability to clear secondary S. pneumoniae
administered 4 days to 6 wk following initial influenza challenge (11, 33, 55). Mice were challenged with 100 PFU
influenza (PR8 strain) administered intranasally, a dose sufficient to elicit an inflammatory response in the lungs with
characteristic histology of human influenza infections (data not
shown), followed 5 days later by intratracheal S. aureus (107
CFU). As seen in Fig. 1A mice challenged with either influenza
or MRSA alone had 50% mortality. In comparison, mice that
were challenged with influenza before S. aureus infection
displayed 100% mortality. To assess if the difference in mortality was secondary to lung bacterial burden, mice were
100pfu H1N1
107 MRSA
100pfu H1N1+107 MRSA
A
Percent survival
100
80
60
40
20
*
0
B
8
*
6
4
2
0
Days Post Infection
Treatment groups
Fig. 1. Mice with viral challenge before bacterial infection have higher bacterial burden in the lung. A: wild-type (WT) mice were challenged with 107
colony-forming units (CFU) methicillin-resistant Staphylococcus aureus (MRSA) either alone or 5 days after infection with 100 PFU H1N1, lungs were harvested
at 24 h, and CFU were evaluated; n ⫽ 6 mice in each group and experiments were repeated 3 times. *P ⬍ 0.05, compared with MRSA infection alone. B: mice
were challenged intranasally with 100 PFU H1N1 alone or intratracheally with 107 CFU MRSA alone or at day 5 post-H1N1 infection mice were challenged
with 107 CFU MRSA. Survival was assessed for each condition; n ⫽ 8 mice in each group and experiments were repeated 3 times. *P ⬍ 0.01, compared with
the double infection group as measured by log rank test.
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intubation and mechanical ventilation, and underwent BAL. Healthy
volunteers without lung disease who underwent BAL via bronchoscopy served as control subjects. BAL samples were filtered through
sterile gauze to remove noncellular particulate material and mucus.
Samples were then centrifuged (1,000 g for 5 min) after collection
to separate the cells and supernatant. The supernatant was aliquoted into small volumes (⬍10 ml). All BAL supernatant samples
were stored at ⫺80°C. Cells were resuspended in RPMI and
macrophages were isolated by adherence purification.
Statistical analysis. All of the data are presented as the means ⫾
SE. Significance was tested by unpaired t-test (for two means) or
one-way ANOVA (for multiple data groups) followed by Tukey post
hoc test. Mouse survival data were analyzed by log-rank test using the
Graph Pad Prism software package.
challenged with either MRSA alone or were infected with
H1N1 as described above and challenged with MRSA. Lungs
were harvested 24 h postbacterial challenge and CFU were
quantitated. WT mice when challenged with bacteria alone
were efficient in clearing the infection. In contrast, mice that
were challenged with MRSA after H1N1 had significantly
higher bacterial burden (2 log) in the lung (Fig. 1B) compared
with mice infected with bacteria alone. We did not find any
systemic dissemination of bacteria in blood or in spleen (data
not shown).
Mice with dual infection have increased IFN␥ expression in
lungs, compared with single infection. Sun and Metzger (55)
have shown that the effect of elevated IFN␥ on antibacterial
immune response to S. pneumoniae post-H1N1 is to impair
phagocytosis by macrophages. To assess IFN␥ expression in
our model, mice were infected with H1N1 intranasally and on
day 5 challenged with 107 MRSA intratracheally. Lungs were
harvested at 24 h, and IFN␥ was expression measured by
real-time RT-PCR. Mice either postviral or postbacterial challenge alone had a fivefold increase in expression of IFN␥
mRNA as compared with uninfected controls (Fig. 2A). Interestingly, challenge with virus and bacteria sequentially resulted
in a significantly greater increase in expression of IFN␥ (10fold). We also observed a significant increase in IFN␥ protein
levels in whole lung (Fig. 2B), most prominent in the lungs of
dual-infected animals.
To determine if this increase in IFN␥ was causally linked to
impaired antibacterial immunity in the lungs of influenzainfected mice, WT and IFN␥⫺/⫺ mice were infected with
H1N1 and on day 5 challenged with MRSA and the lungs were
harvested at 24 h postbacterial challenge for CFU determination. As seen in Fig. 2C, IFN␥⫺/⫺ mice had significantly lower
bacterial burden in the lung (9.6-fold) compared with WT mice
postsequential infection. To assess if this protection was specifically mediated by blunted IFN␥⫺/⫺ responses post-H1N1
infection, WT mice were treated with control antibody or a
neutralizing Ab against murine IFN␥ intraperitoneally (Fig.
2C). Mice treated with control antibody succumbed to bacterial
pneumonia by day 4 (Fig. 2D), which was associated with high
bacterial burden in the lung, whereas mice treated with IFN␥
Lung CFU (log) 24 hrs
post MRSA
Table 1. Demographic characteristics of patients
miR-155 IMPAIRS POSTVIRAL BACTERIAL IMMUNE RESPONSE
B
*
20
*
*
10
5
0
2000
**
1000
0
Ctl
H1N1
MRSA
H1N1/MRSA
Ctl
Treatment groups
H1N1
MRSA
D
6.0
ctl
Percent survival
100
5.5
5.0
*
*
4.5
H1N1/MRSA
Treatment groups
C
4.0
3.5
IFNγ Ab
80
60
40
20
0
IFNγ-/-
CtlAb
anti-IFNγ
0
2
4
8
innate immune responses to viral and bacterial challenge (18,
47). To further investigate if microRNA were contributing to
increased host susceptibility to secondary bacterial challenge,
we measured the expression of miR-155 in whole lung
(Fig. 3A) and lung macrophages (Fig. 3B) harvested from
control and infected mice. miR-155 has been shown to upregulate IFN␥ and has been implicated in innate responses mediated by NF-␬B (64). Infection with either H1N1 or MRSA
B
8
***
6
4
*
2
0
H1N1
MRSA
H1N1/MRSA
Fold increase of miR 155
mRNA in lung macrophages
A
*
*
**
10
5
Control
8
*
6
4
2
0
Untreated
*
*
H1N1
MRSA
0
C
Fold induction of miR 155
mRNA from macrophage
Uninfected
6
Time
Treatment groups
Fold expression of
miR 155 in whole lung
**
*
15
antibody had a more prolonged survival (Fig. 2D) and had
significantly lower bacterial burden when compared infected
mice treated with control Ab (Fig. 2C), indicating that IFN␥
plays an important role in postinfluenza suppression of MRSA
immunity.
Sequential challenge with influenza and MRSA results in
upregulated miR-155 levels in lung and lung macrophages.
MicroRNAs have been recently described to play a role in
Fig. 3. Macrophages harvested from mice postdual infection have higher expression of miR-155
RNA compared with viral or bacterial infection
alone, which is attenuated by anti-IFN␥ Ab treatment. A: WT mice were challenged with 107
CFU MRSA either alone or 5 days after infection
with 100 PFU H1N1, lungs were harvested at 24
h, and expression of miR-155 was measured by
RT-PCR. ***P ⬍ 0.001. B: lung macrophages
were harvested 24 h postbacterial challenge by
collagenase digest and adherence purification and
miR-155 was expression measured by real-time
RT-PCR. C: WT mice were infected intranasally
with 100 PFU H1N1 and on day 5 mice were
inoculated with either control antibody or 80 ␮l
anti-IFN␥ antibody intraperitoneally and 6 h later
mice were challenged intratracheally with 107
CFU MRSA. Lung macrophages were harvested
and expression of miR-155 measured by realtime PCR. *P ⬍ 0.05, **P ⬍ 0.01; n ⫽ 4 in each
group, and experiment was repeated 3 times.
**
3000
IFNγ (pg/ml)
Relative expression of
IFNγ mRNA in whole lung
A
Ctl Ab
anti-IFNγγ
Treatment groups
AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00224.2015 • www.ajplung.org
H1N1/MRSA
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Fig. 2. Dual-infected animals have higher IFN␥ in
lung compared with either challenge alone.
IFN␥⫺/⫺ mice and mice treated with anti-IFN␥
antibody have lower lung bacterial burden compared with WT mice postsequential infection. Mice
were infected with 100 PFU H1N1 or 107 CFU
MRSA alone or sequentially, and IFN ␥ levels in
lung digests were measured by real-time RT-PCR
(A) ELISA (B); n ⫽ 5 mice in each group, experiments repeated twice. *P ⬍ 0.05, **P ⬍ 0.01,
compared with untreated controls. C: WT mice and
IFN␥⫺/⫺ were infected with 100 PFU H1N1 intranasally and on day 5 challenged with 107 CFU
MRSA, lungs were harvested, and bacterial burden
was quantitated. WT mice were also treated with 80
␮l anti-IFN ␥ Ab intraperitoneally or control antibody intraperitoneally 6 h before challenge with
intratracheally 107 CFU MRSA and on day 5 postH1N1 intranasally infection, lungs were harvested
24 h postbacterial challenge, and CFU was quantitated; n ⫽ 5 mice in each group and experiments
were repeated 2 times. **P ⬍ 0.01, as compared
mice infected WT mice. D: WT mice were also
treated with 80 ␮l anti-IFN␥ Ab intraperitoneally or
control antibody intraperitoneally 6 h before challenge with intratracheal 107 CFU MRSA and on
day 5 post-H1N1 intranasal infection and survival
was assessed.
Lung CFU (log) 24hrs
post H1N1/MRSA
L468
miR-155 IMPAIRS POSTVIRAL BACTERIAL IMMUNE RESPONSE
*
SA
Fig. 4. Decreased expression of IL-23 and IL-17
in lungs postsequential infection. WT mice were
challenged with 107 CFU MRSA either alone or 5
days after infection with 100 PFU H1N1, lungs
were harvested at 24 h, and IL-17 (A) and IL-23
(B) gene expression was measured by RT-PCR.
*P ⬍ 0.05; n ⫽ 4 in each group, and experiments
were repeated 3 times.
H
1N
1
+
M
M
R
R
1
tr
o
on
C
R
H
1N
1
+
M
M
Treatment groups
SA
0.0
SA
SA
R
1
1N
H
on
tr
ol
0
0.5
1N
2
1.0
H
4
1.5
l
Relative expression of
IL-17 mRNA in whole lung
*
6
C
Relative expression of
IL-23p19 mRNA in whole lung
B
8
Treatment groups
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tected, with 40% mice alive at day 10 postbacterial challenge
(Fig. 5A). No mice died after 10 days. To understand if
miR-155 was altering the host response to viral challenge or if
the absence of miR-155 had an effect on viral clearance, we
measured viral replication in whole lung by RT-PCR. As seen
in Fig. 5B, there was no significant difference in viral gene
expression between WT and miR 155⫺/⫺ mice, suggesting that
the survival benefit seen in the mutant mice was not secondary
to different levels of viral burden between the two groups.
To further understand the impact of miR-155 on antibacterial responses, WT and miR-155⫺/⫺ mice were infected with
either MRSA alone or sequentially infected and bacterial CFU
were determined 24 h post-MRSA challenge. There was no
difference in lung bacterial burden between WT and miR155⫺/⫺ mice post-MRSA challenge alone (data not shown).
Interestingly, miR-155⫺/⫺ mice displayed significantly lower
lung bacterial burden, compared with WT mice, after infection
with H1N1 and MRSA (Fig. 5C), indicating that miR-155 was
mediating increased mortality and impaired bacterial clearance
postinfluenza pneumonia.
miR-155 negatively regulates IL-17 and IL-23 expression in
postinfluenza pneumonia. We next performed experiments to
determine if improved bacterial clearance observed in dualinfected miR-155⫺/⫺ mice was attributable to more robust
production of IL-17 cytokines. WT and miR-155⫺/⫺ mice were
dually infected as described above, and lungs were harvested
24 h postbacterial challenge. IL-23 and IL-17 mRNA expression was measured by real-time RT-PCR. As previously observed, WT mice displayed reduced expression of IL-23 and
IL-17. In contrast, dually infected miR-155⫺/⫺ mice displayed
two- and fourfold higher expression of IL-23 and IL-17,
respectively, compared with WT animals (Fig. 5, D and E,
respectively), suggesting that the improved clearance in miR155⫺/⫺ mice was partially attributable to greater IL-17 responses in the lung postinfection.
To further identify the cellular source of IL-23, macrophages were harvested from infected WT and miR-155⫺/⫺
mice (as described in METHODS) 24 h postintracheal challenge with MRSA in H1N1-infected mice and expression
was measured by real-time PCR. As shown in Fig. 5F,
macrophages harvested from WT mice postdual challenge
did not have any significant upregulation in IL-23 expression compared with uninfected macrophages. In contrast,
macrophages purified from miR-155⫺/⫺ mice postdual in-
alone resulted in an approximate fivefold increase in miR-155
expression, compared with untreated controls in lung macrophages. By comparison, mice challenged with both H1N1 and
MRSA had significantly higher (9.5-fold higher) expression
than untreated controls in lung macrophages. miR-146 has also
been described to be activated via the NF-␬B pathway and has
been shown to be important for host defense (56). To determine whether the elevation of miR-155 was a selective response or if other microRNA were also involved, we measured
miR-146 levels in whole lung and found no difference in
expression of miR-146 in mice challenged with MRSA alone
or after dual infection (data not shown). This suggests that the
induction of miR-155 was specific in our model.
To assess the role of IFN␥ in the upregulation of miR-155,
lungs were harvested from mice coinfected with H1N1 and
MRSA in the presence or absence of anti-IFN␥ antibody
intraperitoneally. Similar to the results in Fig. 3A, there was an
approximate sixfold increase in expression of miR-155 in
lungs harvested from mice post-H1N1 and post-MRSA dual
infection treated with control Ab. This induction was significantly reduced in mice that received anti-IFN␥ antibody,
indicating a direct in vivo role of IFN␥ in upregulation of
miR-155 (Fig. 3C).
Mice postdual infection have reduced levels of IL-17 and
IL-23. To further understand the mechanism by which influenza suppressed the antibacterial host response, and how this
process might be regulated by miR-155, we measured cytokines that are important for lung antibacterial host defense.
While there was no difference in protein levels of IL-12 or
TNF-␣ in dual-infected mice (data not shown), we found that
sequential infection resulted in significantly blunted IL-23
(Fig. 4A) and IL-17 (Fig. 4B) expression as measured by
real-time RT-PCR, and compared with mice infected with
H1N1 or MRSA alone, suggesting that preceding H1N1 infection attenuates induction of IL-17 and an IL-17-inducing cytokine, IL-23.
miR-155 mediates increased mortality and impaired bacterial clearance in postinfluenza pneumonia. Given that we
observed a significant upregulation of miR-155 in coinfected
mice with impaired antibacterial immunity, WT and miR155⫺/⫺ mice were infected with 100 PFU H1N1 and then
challenged with 107 CFU MRSA on day 5 as described
previously. WT mice had 100% mortality by day 4. By comparison, dually infected miR-155⫺/⫺ mice were partially pro-
A
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miR-155 IMPAIRS POSTVIRAL BACTERIAL IMMUNE RESPONSE
A
B
C
8.0 × 105
WT
miR155-/-
80
60
40
20
*
0
0
2
4
6
Lung CFU 24hrs
post H1N1/MRSA
H1N1
viral gene expression
Percent survival
100
1000
800
600
400
200
6.0 × 105
4.0 × 105
**
2.0 × 105
0
0
miR155-/-
WT
8
Treatment groups
Days Post Infection
WT
*
miR155-/4
2
0
Untreated
H1N1/MRSA
8
Fold expression of IL-23p19
by macrophages
6
F
Relative expression of
IL-17 mRNA in whole lung
Relative expression of
IL-23p19 mRNA in whole lung
E
WT
miR155-/-
*
6
4
2
0
Untreated
Treatment groups
H1N1/MRSA
15
WT
miR155-/-
* **
10
5
0
Untreated
Treatment groups
H1N1 + MRSA
Treatment groups
Fig. 5. MiR-155⫺/⫺ mice have decreased bacterial burden in the lung and increased expression of IL-23 and IL-17 compared with WT mice. A: WT and
miR-155⫺/⫺ mice were challenged intranasally with 100 PFU H1N1 and on day 5 post-H1N1 infection challenged with 107 CFU MRSA and survival was
assessed. WT and miR-155⫺/⫺ mice were challenged intranasally with 100 PFU H1N1 and on day 5 post-H1N1 infection challenged with 107 CFU MRSA.
Lungs were harvested at 24 h and viral gene expression was analyzed by RT-PCR (B) and lung bacterial CFU were quantified (C). Lungs were harvested 24
h postbacterial challenge and expression of IL- 23 (D) and IL- 17 (E) levels and measured by real-time PCR. Lung macrophages 24 h postbacterial challenge
from H1N1/MRSA-infected mice or uninfected control mice were harvested by collagenase digest and adherence purification and IL-23p19 and expression was
measured by real-time PCR (F). *P ⬍ 0.05, **P ⬍ 0.01; n ⫽ 4 in each group, and experiments were repeated 3 times.
fection had a robust twofold induction in IL-23 expression
compared with macrophages harvested from infected WT
mice.
Treatment with IL-17 antibody partially abolishes the protective phenotype observed in miR-155⫺/⫺ mice. To establish
that the improved antibacterial host response observed in
miR-155⫺/⫺ mice was secondary to enhanced IL-17 expression, WT and miR-155⫺/⫺ mice were infected with H1N1
intranasally and treated with 100 ␮g anti-IL-17 antibody or
control antibody intraperitonelly 4 h before challenge with
MRSA intratracheally and then assessed for lung bacterial
clearance 24 h later. WT mice treated with the control
antibody had high bacterial burden. As observed previously,
miR-155⫺/⫺ mice treated with control Ab were significantly
more efficient in clearing bacteria. However, as seen in
Fig. 6A administration of anti-IL-17 Ab 4 h before bacterial
challenge partially mitigated the improved bacterial clearance
observed in miR-155⫺/⫺ mice.
To provide further evidence that IL-23, which is upstream of
IL-17 and is produced more abundantly by macrophages post-
WT
A
Lung CFU 24hrs
post H1N1/MRSA
6.0 × 105
mirR155-/- + antiIL-17
*
4.0 × 105
2.0 × 105
*
B
6.0 × 105
Lung CFU 24hrs
post H1N1/MRSA
miR155-/- Control Ab
4.0 × 105
*
2.0 × 105
0
0
WT
Treatment groups
WT + rm IL-23
Treatment groups
Fig. 6. miR-155⫺/⫺ mice treated with anti-IL-17 antibody before bacterial challenge have higher bacterial burden in the lung. A: WT and miR-155⫺/⫺ mice were
challenged intranasally with 100 PFU H1N1 and on day 5 post-H1N1 infection challenged with 107 CFU MRSA. Four hours before bacterial challenge
miR-155⫺/⫺ mice were treated with either a control antibody or 100 ␮g of anti-IL-17 antibody intraperitoneally. Lungs were harvested at 24 h and CFU were
quantified. B: WT mice were infected with H1N1 and 4 h before challenge with MRSA given vehicle control or rmIL-23 intratracheally Lungs were harvested
at 24 h and CFU quantified. *P ⬍ 0.05; n ⫽ 5 in each group, and experiments were repeated 2 times.
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D
miR155-/-
WT
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miR-155 IMPAIRS POSTVIRAL BACTERIAL IMMUNE RESPONSE
miR155 antagomir
Lung CFU 24hrs
post H1N1/MRSA
A
Control antagomir
5
8.0 × 10
**
6.0 × 105
4.0 × 105
2.0 × 105
0
Treatment groups
Uninfected WT
C
20
WT H1N1/MRSA + Antagomir 155
*
15
10
5
0
Treatment groups
6
**
4
2
0
Treatment groups
dual infection in miR-155⫺/⫺ mice was playing a mechanistic
role in our model, WT mice were infected with H1N1 and 4 h
before MRSA intratracheally reconstituted with either vehicle
control or rmIL-23. Lungs were harvested 24 h after bacterial
challenge and CFU were quantitated. As seen in Fig. 6B, mice
treated with rmIL-23 had significantly lower bacterial burden
in the lung compared with vehicle-treated WT mice postsequential infection.
Treatment with miR-155 antagomir improves bacterial
clearance and IL-17 expression in dual-infected animals. To
evaluate a therapeutic potential for miR-155 antagonism,
WT mice were infected with H1N1 intranasally, and then 4
h before challenge with intratracheal MRSA, they were
treated with either sham or miR-155 antagomir intraperitoneally. Lungs were harvested 24 h after bacterial challenge,
and bacterial load was quantitated. Dually infected mice
treated with sham antagomir had high lung MRSA CFU
whereas treatment with miR-155 antagomir reduced lung
MRSA CFU (Fig. 7A) without having any effect on viral
titres in the lung (data not shown). Moreover, treatment of
H1N1/MRSA-infected mice with miR-155 antagomir resulted in significantly increased expression of both IL-23
and IL-17 (Fig. 7, B and C, respectively). Collectively, these
data indicate that antagonizing miR-155 offers protection in
postinfluenza bacterial pneumonia.
Alveolar macrophages from dual-infected patients have
higher miR-155 levels. To determine whether observations
made in the murine model were clinically relevant in patients with influenza pneumonia, we examined expression of
miR-155 levels in patients admitted to our ICU with ARDS
from bacterial pneumonia postinfluenza A infection. Patient
characteristics at study entry are shown in Table 1. Intubated patients that tested positive for influenza A (H1N1)
and had tracheal aspirate or sputum positive for Grampositive bacteria were selected. Healthy volunteers without
lung disease who underwent BAL via bronchoscopy served
as control subjects. BAL from 12 patients with secondary
bacterial pneumonia was harvested, and macrophages were
purified by adherence purification with a purity of 96% as
determined by flow cytometry. miR-155 expression was
measured by real-time RT-PCR. As shown in Fig. 8, macrophages harvested from patients with secondary bacterial
pneumonia due to either MRSA or S. pneumoniae demonRelative expression of
miR155 mRNA by human macrophages
WT H1N1/MRSA + Control
Antagomir
Relative expression of
IL-17 mRNA in whole lung
Relative expression of
IL-23p19 mRNA in whole lung
B
*
6
*
4
2
0
Control
H1N1/MRSA
H1N1/S.pneumo
Treatment groups
Fig. 8. Human alveolar macrophages purified from patients with H1N1 and
bacterial coinfection had increased expression of miR-155. Alveolar macrophages were purified by adherence purification from bronchoalveolar lavage
(BAL) obtained from healthy subjects who underwent bronchoscopy for
research purposes or patients admitted to our intensive care unit with respiratory failure from secondary bacterial pneumonia post H1N1, and expression of
miR-155 was measured by real-time PCR. *P ⬍ 0.05; n ⫽ 12 patients and
n ⫽ 3 controls.
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Fig. 7. Treatment with miR-155 antagomir decreased bacterial burden in the lung and upregulates expression of IL-23 and IL-17. WT mice
were challenged intranasally with 100 PFU
H1N1 and on day 5 post-H1N1 infection challenged with 107 CFU MRSA and treated with
sham antagomir or miR-155 antagomir intranasally. Lungs were harvested at 24 h and CFU
were quantified (A). Cytokine expression of
IL-23 and IL-17 were measured in lungs by
real-time RT-PCR (B and C). *P ⬍ 0.05, **P ⬍
0.01; n ⫽ 5 in each group, and experiments were
repeated 2 times.
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miR-155 IMPAIRS POSTVIRAL BACTERIAL IMMUNE RESPONSE
strated significantly higher miR-155 expression (4.5- and
3.1-fold, respectively) compared with control macrophages
harvested from healthy subjects.
DISCUSSION
AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00224.2015 • www.ajplung.org
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Our findings demonstrate a novel molecular mechanism by
which influenza A impairs host defense against secondary
MRSA infection. The majority of severe and fatal influenza
infections are related to secondary bacterial pneumonia (9)
highlighting the importance of understanding compromised
immune defense in this context. Furthermore, recent findings
during the recent H1N1 pandemic have shown a predilection
for secondary CA-MRSA infections in fatal influenza A (25).
We found that miR-155 is induced by influenza and this
microRNA inhibits protective IL-23 and IL-17 responses. Antagonizing miR-155 postinfluenza A infection increased IL-23
and IL-17 expression and improved the immune response to
secondary S. aureus challenge. These observations may have
implications for the attenuation of host defense against a
number of pathogens, as the IL-17 pathway has been implicated in host immunity against bacterial and fungal pathogens.
The observation that influenza A increases susceptibility to
S. aureus was reported many years ago (23); however, the
molecular mechanisms for this effect have remained elusive.
Furthermore, pathologic synergism between influenza A and S.
aureus products has been identified (58, 59, 70). Recently,
influenza A was shown to exacerbate secondary S. aureus
infection in mice as measured by decreased bacterial clearance
and increased mortality (54). In the aforementioned study,
influenza was shown to inhibit NK cell production of TNF-␣,
which resulted in impaired antimicrobial function of macrophages. In our S. aureus model, WT and miR-155⫺/⫺ mice
displayed no difference in TNF-␣ production in the lung
induced by S. aureus at 24 h postchallenge, which was significantly upregulated compared with that in control mice despite
worsened clearance of S. aureus in WT mice (data not shown).
miR-155 is known to affect TNF-␣ stabilization and increases
its translation (63); however, we did not see changes in TNF-␣
in our current studies.
Sun and Metzger (55) have shown influenza induces IFN␥
expression, which increases susceptibility to streptococcal
pneumonia. Similarly, we observed increased production of
IFN␥ in mice with dual infection. Our study indicates that
IFN␥ drives enhanced miR-155 expression, which mediates
impaired innate immune response to bacterial challenge. Sun
and Metzger proposed the mechanism of impaired bacterial
clearance to be secondary to impaired phagocytosis of bacteria
by macrophages, due to reduced expression of the MARCO
scavenger receptor. Scavenger receptors may be relevant in our
model as well since macrophages transfected with antagomir to
miR-155 demonstrate increased ability to phagocytize S. aureus due to upregulation of scavenger receptor SRA (12). We
have not directly measured the phagocytic ability of alveolar
macrophages from dual-infected mice to engulf MRSA, but it
is possible that it would be enhanced given our previous
results; thus the defect in vivo likely relates to impaired
clearance (12). Type 1 interferons also have been implicated in
increased susceptibility to secondary bacterial pneumonia (41,
50) although we did not find any difference in IFN␣/␤ levels in
our model (data not shown).
IL-17 has been implicated in immunity against bacterial
pathogens including S. aureus (27). In fact, IL-17⫺/⫺ mice had
increased bacterial burden in the lung in mice challenged with
H1N1 and subsequently with S. aureus. These mice were
shown to have increased inflammation and decreased clearance
of bacteria associated with substantially decreased IL-17, IL22, and IL-23 production after S. aureus infection (27). In
addition, they demonstrated that the majority of IL-17A-producing cells were CD4⫹ or ␥␦ T cells, which were significantly
decreased when bacterial infection was preceded by viral
challenge. Furthermore, overexpression of IL-23 in influenza
A, S. aureus-coinfected WT mice rescued the induction of
IL-17 and IL-22 and markedly improved bacterial clearance.
This is consistent with what we observed in our model, and our
work now links these changes in IL-17 and IL-23 to upregulation of miR-155.
Many studies have demonstrated the critical role of macrophages in antibacterial host defense and specifically in postviral secondary bacterial pneumonia (4, 17, 55). IL-23 has been
described to be involved in the clearance of infectious pathogens, immune responses against malignancies, and development of autoimmune diseases (8, 28, 35). CD11c⫹ cells have
been shown to be primary producers of IL-23 in the lungs
postinfection or injury, including CD11c⫹ macrophages/monocytes (3). IL-23 has been implicated in Th17 cell regulation,
proliferation, and cytokine production (34, 68). In addition to
CD4⫹ T cells, many innate immune cells respond to IL-23 and
are important in resistance to infection. We have shown sequentially infected mice have significantly low expression of
IL-23 in lungs, and reconstitution of IL-23 intratracheally
improves the ability of WT mice challenged with H1N1 and
MRSA to clear bacteria. We postulate that rmIL-23 has a dual
role in offering protection: 1) IL-23 potentiates production of
IL-17 as well as IL-22, and both these cytokines have been
shown to offer protection against secondary bacterial pneumonia postinfluenza infection (22, 27); and 2) IL-23 has also been
shown to suppress IL-12-mediated potentiation of IFN-␥ production, thus offering additional protection against secondary
bacterial pneumonia (53).
miR-155 has been shown to be upregulated by various
TLR ligands in macrophages through either the MyD88 or
TRIF signaling pathway (45). In mouse models of autoimmune diseases like experimental allergic encephalomyelitis
and arthritis, it has been shown that miR-155-deficient
animals have defects in skewing toward the Th17 lineage
both in vivo and in vitro (40, 43, 69). Contrary to the
autoimmune models, in our study we see upregulation of
miR-155 in lung, specifically by macrophages postviral and
bacterial challenge associated with significantly blunted
IL-23 response, which likely results in decreased IL-17 seen
in lungs postdual infection.
In our study, we noted increased IFN-␥ levels postdual
challenge with virus and bacteria and IFN-␥ has been shown
previously to regulate miR-155 expression (2). Moreover,
IFN-␥ has also been shown to negatively regulate IL-23 in
murine macrophages by histone modification in a model of
murine experimental colitis (52). We speculate that the mechanism of decreased IL-23 secretion by lung macrophage is
mediated via IFN-␥ signaling upregulating miR ⫺155 in macrophages and potentially resulting in histone acetylation. miR155⫺/⫺ mice are protected with improved bacterial clearance
miR-155 IMPAIRS POSTVIRAL BACTERIAL IMMUNE RESPONSE
GRANTS
This study was supported by National Heart, Lung, and Blood Institute
Grants HL-119682 and HL-123515 and National Institute of Allergy and
Infectious Diseases Grant AI-117229.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the
author(s).
AUTHOR CONTRIBUTIONS
A.P., R.E., P.K.P., and U.B. performed experiments; T.J.S., B.B.M., and
U.B. interpreted results of experiments; T.J.S., B.B.M., and U.B. edited and
revised manuscript; M.N.B., S.L.K., and U.B. conception and design of
research; U.B. analyzed data; U.B. prepared figures; U.B. drafted manuscript;
U.B. approved final version of manuscript.
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