Nonhematopoietic Cells Are Key Players in
Innate Control of Bacterial Airway Infection
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J Immunol 2011; 186:3130-3137; Prepublished online 26
January 2011;
doi: 10.4049/jimmunol.1003565
http://www.jimmunol.org/content/186/5/3130
http://www.jimmunol.org/content/suppl/2011/01/26/jimmunol.100356
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Supplementary
Material
Salomé LeibundGut-Landmann, Kerstin Weidner, Hubert
Hilbi and Annette Oxenius
The Journal of Immunology
Nonhematopoietic Cells Are Key Players in Innate Control of
Bacterial Airway Infection
Salomé LeibundGut-Landmann,* Kerstin Weidner,* Hubert Hilbi,† and Annette Oxenius*
M
ucosal surfaces are the entry sites of airborne and foodborne pathogens, which overall constitute a major
continuous microbial challenge for the host. Mucosal
tissues have adapted to confront this assault by various means:
during steady-state conditions (i.e., in absence of pathogenic
microbes), epithelial cells, together with their mucous lining and
the constitutive expression of antimicrobial substances, represent
a physical and biological barrier for commensal microorganisms.
The innate defense system in the lung consists of: 1) airway epithelial cells (AECs) and their products (the airway surface liquid,
antimicrobial substances, cytokines, and chemokines); 2) alveolar
macrophages (AMs), which are strategically positioned at the
luminal surface of the epithelial lining being responsible for
efficient phagocytic uptake and clearance of microbes; and 3)
dendritic cells (DCs), which were shown to capture particulate Ags
through transepithelial luminal dendrites (1–3).
Infections with pathogens in the lung lead to the induction of
a local inflammatory response. The fact that AECs outnumber AMs
*Institute of Microbiology, Swiss Federal Institute of Technology Zurich, 8093
Zurich, Switzerland; and †Ludwig-Maximilians-University Munich, Max von
Pettenkofer-Institute, 80336 Munich, Germany
Received for publication October 26, 2010. Accepted for publication December 15,
2010.
This work was supported by the Heuberg-Foundation, ETH Zurich, and Swiss National Science Foundation (Grant 310000-113947).
S.L.-L. and A.O. designed the research; S.L.-L. and K.W. performed the experiments;
S.L.-L., K.W., and A.O. analyzed the data; and S.L.-L., A.O., and H.H. wrote the
article.
Address correspondence and reprint requests to Dr. Annette Oxenius, Institute of
Microbiology, ETH Zürich, Wolfgang-Pauli-Strasse 10, HCI G401, 8093 Zürich,
Switzerland. E-mail address: [email protected]
The online version of this article contains supplemental material.
Abbreviations used in this article: AEC, airway epithelial cell; AM, alveolar macrophage; BAL, bronchoalveolar lavage; BMDM, bone marrow-derived macrophage;
DC, dendritic cell; IL-1Ra, IL-1R antagonist; imAEC, long-term culture of primary
alveolar epithelial cell; PAMP, pathogen-associated molecular pattern; PRR, pattern
recognition receptor; T4SS, type IV secretion system.
Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1003565
by far renders their involvement in initiating or amplifying the
innate immune response very likely. Indeed, a number of studies
have shown that AECs can express a variety of membrane-bound or
cytosolic pattern recognition receptors (PRRs) and are directly
responsive to pathogen-associated molecular patterns (PAMPs) (1,
3, 4). However, most studies have analyzed either isolated primary
AECs or lung epithelial cell lines in in vitro experiments, and it
remains unclear whether AECs respond comparably in the intact
lung tissue in presence of airway surface liquid and AMs, which
are more broadly equipped with PRRs. Moreover, it is conceivable
that there is significant cross talk between AECs and AMs during
an inflammatory response after airway infection with pathogenic
bacteria.
We have used an airway infection model with the Gram-negative
bacterium Legionella pneumophila, the causative agent of Legionnaire’s disease, to assess in vivo the importance of nonhematopoietic cells in the lung and their communication with
AMs to initiate an inflammatory response, which, in turn, is
critical for rapid control of L. pneumophila infection (5–9). On
airway infection, L. pneumophila infect AMs, and in contrast to
extracellular bacteria, L. pneumophila resist intracellular lysosomal degradation by means of their icm/dot type IV secretion
system (T4SS) (10). Within their host cells, L. pneumophila establish and reside within a “Legionella-containing vacuole” that is
permissive for intracellular multiplication (11). Hence the induction of an inflammatory response that recruits other immune
cells to the site of infection is necessary to eventually control this
pulmonary bacterial infection.
We and others have previously shown that in vivo L. pneumophila-induced inflammation critically depends on both MyD88
signaling on the host side and on a functional T4SS on the bacterial side (6–9, 12). This is in contrast with in vitro experiments
with isolated cells where the L. pneumophila T4SS plays only
a minor role for the production of various proinflammatory
cytokines (13). This apparent discrepancy between in vitro and
in vivo experiments may be caused by the involvement of various
cell subsets of hematopoietic and nonhematopoietic origin par-
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Airborne pathogens encounter several hurdles during host invasion, including alveolar macrophages (AMs) and airway epithelial
cells (AECs) and their products. Although growing evidence indicates pathogen-sensing capacities of epithelial cells, the relative
contribution of hematopoietic versus nonhematopoietic cells in the induction of an inflammatory response and their possible interplay is still poorly defined in vivo in the context of infections with pathogenic microorganisms. In this study, we show that nonhematopoietic cells, including AECs, are critical players in the inflammatory process induced upon airway infection with Legionella
pneumophila, and that they are essential for control of bacterial infections. Lung parenchymal cells, including AECs, are not
infected themselves by L. pneumophila in vivo but rather act as sensors and amplifiers of inflammatory cues delivered by L.
pneumophila-infected AM. We identified AM-derived IL-1b as the critical mediator to induce chemokine production in nonhematopoietic cells in the lung, resulting in swift and robust recruitment of infection-controlling neutrophils into the airways.
These data add a new level of complexity to the coordination of the innate immune response to L. pneumophila and illustrate how
the cross talk between leukocytes and nonhematopoietic cells contributes to efficient host protection. The Journal of Immunology,
2011, 186: 3130–3137.
The Journal of Immunology
Materials and Methods
Mice, bacteria, and infections
C57BL/6, myd882/2 (15), il-1r2/2 (16), caspase-12/2 (17), H-2Kb-tsA58
(18), and H-2Kb-tsA58 3 myd882/2 mice were bred at the ETH Zürich or
purchased from Janvier Elevage and used at 7–16 wk of age (age-matched
within experiments). All mice were backcrossed on C57BL/6 except the H2Kb-tsA58 transgenic mice, which were on a mixed Sv129 3 C57BL/6
background. All animal experiments were performed in accordance with
institutional policies and have been reviewed by the cantonal veterinary
office. For generation of chimeric mice, C57BL/6, myd882/2, il-1r2/2, and
caspase-12/2 mice were gamma-irradiated (950 Rad) and reconstituted
with 1 3 106 bone marrow cells derived from C57BL/6 or myd882/2 mice.
Experiments were performed after at least 8 wk of reconstitution. The
chimerism was checked in some control animals, and it was verified, in
particular, that the hematopoietic compartment was of donor origin (95%
reconstitution in the hematopoietic compartment and .85% in AMs; data
not shown). Normal cytokine production (IL-12p40, Supplemental Fig. 1;
IL-1b, Fig. 5C) in B6 . MyD882/2 chimeras further proved, on a functional level, that engraftment had taken place.
The L. pneumophila strains used in this study were JR32 (wild type
Philadelphia-1) (19), JR32-GFP (20), GS3011 (DT, icmT deletion mutant
lacking a functional Icm/Dot T4SS) (21), and DT-GFP (20). L. pneumophila was grown for 3 d on charcoal yeast extract agar plates. Liquid
cultures were inoculated in ACES yeast extract medium at an OD600 of 0.1
and grown for 21 h at 37˚C. Chloramphenicol (5 mg/ml) was added to
maintain plasmids.
Mice were infected intranasally with 5 3 106 CFU L. pneumophila
(grown on plates) in 20 ml prewarmed PBS that were applied directly onto
the nostril of anesthetized mice using a Gilson pipette. Bronchoalveolar
lavage (BAL) was collected by flushing the airways with 1 ml PBS after
sublethal anesthesia of mice with xylazine (0.38 mg), ketamine (1.9 mg),
and acepromazine (57 mg), and perfusion of the blood circuit with PBS.
Cells were recovered from the BAL by centrifugation, and cell-cleared
BAL was stored at 220˚C for further analyses. CFU counts were determined by plating dilutions of BAL and homogenized lung tissue onto
charcoal yeast extract agar plates.
Cells
Bone marrow-derived macrophages (BMDM) were generated by plating
bone marrow in complete medium (RPMI 1640, 10% FCS, 2 mM glutamine, nonessential amino acids, 50 mM 2-ME, 1 mM sodium pyruvate, 10
mM HEPES and antibiotics) supplemented with 20% L929-cell conditioned medium. On day 4 of culture, the cells were further supplemented
with complete BMDM medium. After a total of 6–7 d of culture, the adherent macrophages were harvested and seeded in 96-well plates at 1 3
105 cells/well, and 1 d later, cells were infected with the indicated multiplicity of infection of L. pneumophila from liquid cultures. Infection was
synchronized by centrifugation, and the infected cells were incubated at
37˚C for 6 h (for in vitro cross-talk experiments and protein measurements), or for 3 h in case of mRNA analysis.
Long-term cultures of primary alveolar epithelial cells (imAECs) were
isolated from lungs of H-2Kb-tsA58 and H-2Kb-tsA58 3 myd882/2 mice by
FACS, based on their CD452 CD312 podoplanin+ phenotype. Long-term
cultures of these cells were established by culturing the sorted cells in
complete medium supplemented with 100 U/ml IFN-g at 33˚C. IFN-g was
removed and cells were grown at 37˚C during 3 d before experiments, and
cells were seeded in 96-well plates at 5 3 104 cells/well 1 d before stimulation. For in vitro cross-talk experiments, BAL from infected C57BL/6
mice or the supernatant of L. pneumophila-infected BMDM was transferred onto AECs for 15 h, and chemokines were analyzed in the supernatant. To prevent direct infection of AECs by cotransferred bacteria,
we added 10 mg/ml gentamicin to the transferred BMDM supernatants.
IL-1R antagonist (IL-1Ra) was added where indicated.
Reagents
All Abs for FACS and FACS analysis were from BioLegend or BD Biosciences. The podoplanin-specific Ab (clone 8.1.1) was a kind gift from
Prof. Cornelia Halin (ETH, Zurich, Switzerland). The neutrophil-depleting
NimpR14 Ab was a kind gift of Dr. Nancy Hogg (Cancer Research UK,
London, U.K.). One hundred micrograms of Ab was injected i.p. daily from
1 d before infection to 3 d postinfection. A goat anti-mouse anti–IL-1b Ab
from R&D Systems in combination with an HRP-conjugated anti-goat Ab
from Santa Cruz Biotechnology was used for Western blotting according to
standard protocols. IL-1Ra (Kineret/Anakinra; Amgen Europe B.V.) was
used at 1–10 mg/ml. rIFN-g was produced from X63-IFN-g transfectants
(a kind gift from Prof. Hans Hengartner, University of Zurich). Gentamicin
was from AppliChem.
FACS
Lungs were digested for 30 min at 37˚C with Collagenase I and DNase I,
and a single-cell suspension was then prepared by forcing the organ
through a 70-mm strainer. Cells from the BAL and lung were stained by
standard procedures using fluorescence-labeled Abs. Cell populations were
defined as CD45+ CD11c2 F4/802 Ly6C+ Ly6G+ (neutrophils), CD45+
CD11c2 F4/802 Ly6C+ Ly6G2 (inflammatory monocytes), and CD45+
CD11chi F4/80+ autofluorescence+ (AMs).
Cytokine and chemokine measurements
The concentration of CXCL1, CXCL2, CCL2, CCL3, IL-12p40, and TNF-a
in cell culture supernatants and in the BAL was determined by ELISA.
Secreted IL-1b levels were quantified by cytometric bead array (BD
Biosciences) according to the manufacturer’s instructions. IL-1b mRNA
was quantified by real-time PCR using SensiMix Plus SYBR kit (Quantace), a RotorGene 3000 Thermal Cycler (Corbett Research), and the
following primers: IL-1b forward, 59-caaccaacaagtgatattctccatg-39; IL-1b
reverse, 59-gatccacactctccagctgca-39; b-actin forward, 59-ccctgaagtaccccattgaac-39; b-actin reverse, 59-cttttcacggttggccttag-39. Values were normalized with respect to b-actin and were expressed relative to the levels of
uninfected controls.
Statistical analysis
The two-tailed unpaired t test was applied for statistical analysis: *p ,
0.05; **p , 0.01; ***p , 0.001.
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ticipating in a communication network in vivo, with subsetspecific signal requirements. Furthermore, the dual dependence
on the L. pneumophila T4SS and host MyD88 signaling implies
that a combination of cytoplasmic recognition events of T4SSsecreted PAMPs and host cell-receptor signaling events via
MyD88 is required to initiate an inflammatory response in vivo.
TLR2, TLR5, and TLR9 have all been shown to recognize L.
pneumophila. However, the strong dependence of the induction of
inflammation on the adaptor protein MyD88 cannot be explained
solely by the recognition of L. pneumophila through these
receptors. Mice deficient in these receptors or mice infected with
a TLR5 ligand-deficient L. pneumophila strain show only subtle or
no defects in pathogen control (6–9), whereas MyD88-deficient
mice display high bacterial counts in the lung compared with
control mice (6–9, 14). In addition to being required for signaling
pathways stimulated by TLRs, MyD88 is essential for signaling
events activated by the IL-1 family of receptors (15). The different
MyD88-dependent signaling pathways that may be activated
concomitantly during L. pneumophila infection may, however,
function in different cell types. It has been shown recently that
MyD88 is required in cells of the hematopoietic compartment for
efficient control of airway L. pneumophila infection (14). However, the question whether MyD88 may also play a role in nonhematopoietic cells has not been addressed so far.
In this article, we show proof for this hypothesis and unravel an
intricate cross talk between lung-resident nonhematopoietic cells
and AMs: the initial recognition event of L. pneumophila occurs in
AMs that phagocytose L. pneumophila and in response secrete
IL-1b via T4SS-dependent activation of caspase-1. IL-1b is then
sensed by nonhematopoietic cells, including AECs (which by
themselves are not infected in vivo), and triggers MyD88-dependent chemokine production leading to the rapid recruitment
of neutrophils, monocytes, and DCs. Thus, nonhematopoietic cells
were identified as essential amplifiers of lung inflammation, which
actively promote the recruitment of migratory innate immune cells,
rendering them critical contributors to host protection from pathogenic airway infections.
3131
3132
CROSS TALK BETWEEN LUNG PARENCHYMA AND IMMUNE CELLS
Results
L. pneumophila-induced inflammation in the airways is
MyD88 and T4SS dependent
Epithelial cells are greatly abundant among the stromal cells in the
lung and are strategically positioned at the interface between the
luminal airspace and the lung tissue. Given these features of AECs
and their previously described capacity to respond directly to
various microbial stimuli (1, 3, 4) and specifically to L. pneumo-
FIGURE 1. L. pneumophila infection induces a strong infiltration of neutrophils and inflammatory monocytes into the airways. A, C57BL/6 and myd882/2
mice were infected intranasally with L. pneumophila JR32, and neutrophils, monocytes, and AMs were quantified in the BAL 18 h postinfection. Data
show mean and SD of three mice per group and are representative of two independent experiments. B, Neutrophils, monocytes, and AMs were quantified in
the BAL of C57BL/6 mice that were infected intranasally with the L. pneumophila strains JR32 or DT 4 and 28 h previously. Data show mean and SD of
three mice per group and are representative of two independent experiments. C, Chemokine levels were determined by ELISA in the BAL of the mice
shown in B. D, Live bacteria were quantified 4 d postinfection with L. pneumophila JR32 in the lung and BAL of C57BL/6 mice that were either depleted of
neutrophils or left untreated. Each symbol represents one mouse. Data shown are representative of two independent experiments. *p , 0.05, **p , 0.01,
***p , 0.001 (two-tailed unpaired t test).
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In a previous study, we found that L. pneumophila-induced inflammation upon systemic infection is dependent on both the
bacterial T4SS and pathogen recognition via a MyD88-controlled
pathway (8). This finding also holds true for L. pneumophilainduced inflammation in the airways of mice infected via the intranasal route (Fig. 1) (5, 9). We show here that the production of
proinflammatory cytokines was induced, and that large numbers of
leukocytes were recruited into the airways of L. pneumophilainfected mice (Fig. 1A, 1B). Neutrophils could be detected in the
BAL as early as 4 h postinfection, and their numbers increased
with time, whereas inflammatory monocytes and DCs appeared
later (Fig. 1A, 1B, and data not shown). In contrast, AM numbers
in the BAL remained unchanged upon infection (Fig. 1A, 1B). The
infiltration of neutrophils, inflammatory monocytes, and DCs coincided with the production of chemokines in the BAL (Fig. 1C).
Neutrophil-attracting CXCL1 and CXCL2 were detected at the
onset of neutrophil infiltration, whereas the monocyte- and DCattracting chemokines CCL2, CCL3, and CCL20 reached their
maximum levels only at later time points (Fig. 1C and data not
shown). The production of proinflammatory cytokines and chemokines, and the recruitment of inflammatory leukocytes to the
airways in response to infection were dependent on both host
MyD88 (Fig. 1A, Supplemental Fig. 2A) (9) and the bacterial
T4SS (Fig. 1B, 1C, Supplemental Fig. 2B). Infection with L.
pneumophila is controlled rapidly by the host innate immune response (5, 6, 8, 9), and neutrophils may play a critical part.
Consistent with this notion, we show that neutrophil-depleted
mice (neutrophil depletion efficacy shown in Supplemental Fig.
3) had a strong defect in controlling L. pneumophila in the lung
and the BAL (Fig. 1D). The concurrent recognition of L. pneumophila via MyD88-dependent and cytoplasmic mechanisms is
thus essential not only for induction of the inflammatory response
but also for the reduction of the pathogen load and for host protection.
Nonhematopoietic cells contribute critically to L.
pneumophila-induced inflammation and bacterial control
The Journal of Immunology
phila in vitro (22), we next evaluated the role of nonhematopoietic
cells, including AECs, in host protection from L. pneumophila
infection. To this end, we generated chimeric mice, wherein all
nonhematopoietic cells including epithelial cells were deficient of
MyD88, whereas all hematopoietic cells were of wild-type origin.
These chimeric mice showed a strong defect in bacterial control
upon L. pneumophila infection (Fig. 2A), indicating that nonhematopoietic cells, including most likely AECs, indeed are critically involved in controlling L. pneumophila airway infection in
an MyD88-dependent manner. In fact, nonhematopoietic cells
were essential for the early production of chemokines and the
3133
AM-derived IL-1b induces chemokine production in epithelial
cells
FIGURE 2. MyD88 expression in nonhematopoietic cells is essential for
efficient control of L. pneumophila infection. A, Irradiation chimeras were
generated by reconstituting C57BL/6 or myd882/2 mice with C57BL/6
bone marrow. Bacterial counts were determined in the lung and BAL 5 d
postinfection with L. pneumophila strain JR32. Each symbol depicts one
mouse, and the experiment is representative of two independent repeats. B
and C, Chemokine levels (B) and neutrophil counts (C) were determined in
the BAL of infected chimeras 4 h postinfection with L. pneumophila JR32.
Data are mean and SD of three mice per group (one uninfected mouse) and
are representative of three independent experiments. D, C57BL/6 mice
were infected with GFP-expressing L. pneumophila strain JR32 or DT, and
the cells that have taken up bacteria in the lung were identified 4 h postinfection by FACS. GFP+ cells within the scatter-gated population (left
column), CD45 expression on GFP+ cells (middle column), and F4/80+
CD11c+ expression on the CD45+ population (right column). *p , 0.05,
**p , 0.01, ***p , 0.001 (two-tailed unpaired t test).
Our findings that nonhematopoietic cells are not directly infected
in vivo by L. pneumophila is consistent with a model in which they
respond to inflammatory mediators rather than sensing L. pneumophila directly. Given the MyD88 dependence of the response of
the nonhematopoietic cells, we hypothesized that IL-1R– or IL18R–dependent events rather than TLR-mediated signaling may
be involved. To assess the IL-1b and IL-18 responsiveness of
nonhematopoietic cells in the lung, we focused on alveolar type I
cells, which constitute ∼95% of the internal lung surface and are
therefore prominent representatives of nonhematopoietic cells in
the lung. Indeed, CXCL1 production could be induced in vitro in
imAECs by IL-1b (Fig. 3A), but not by IL-18 (data not shown).
Consistent with this finding, IL-18R–deficient mice do not exhibit
defects in control of airway L. pneumophila infection (data not
shown) (7). Furthermore, imAECs did also produce chemokines in
response to stimulation with supernatants from L. pneumophilainfected BMDM (Fig. 3B, 3C) or with BAL of L. pneumophilainfected mice (Fig. 3D). The dependence on IL-1b of this response was demonstrated by the lack of chemokine production in
MyD88-deficient imAECs and by the efficient blocking of the
response in wild type imAECs by the IL-1Ra (Fig. 3C, 3D). Of
note, in vitro L. pneumophila infection of BMDM also resulted in
production of low levels of CXCL1, consistent with previously
published data of L. pneumophila-infected BMDM (23) or
BMDM stimulation with specific TLR ligands (24, 25). However,
the level of CXCL1 production in BMDM was at least one order
of magnitude lower than the amount produced by imAECs stimulated with supernatants from L. pneumophila-infected BMDM
(Fig. 3C and data not shown). The superior in vivo relevance of
chemokine production by nonhematopoietic cells was further
demonstrated by the finding that CXCL1 and CXCL2 contents in
the BAL of L. pneumophila-infected chimeric mice harboring wild
type hematopoietic cells (including AMs) in hosts with selective
MyD88 or IL-1R deficiency in nonhematopoietic cells were severely reduced (Figs. 2B, 4A).
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swift attraction of neutrophils to the airspace of the lung (Fig. 2B,
2C). Importantly, these results do not exclude a comparably critical role of MyD88 signaling in hematopoietic cells for control of
airway L. pneumophila infection, as has been previously shown in
chimeric mice lacking MyD88 selectively within hematopoietic
cells (14) (data not shown).
Nonhematopoietic cells including AECs could produce chemokines either in response to direct recognition of L. pneumophila
or in response to inflammatory mediators released by other cells
such as L. pneumophila-infected AMs. In support of the first hypothesis, lung epithelial cell lines and imAECs could be infected
by L. pneumophila and produced chemokines in response to L.
pneumophila infection in vitro (unpublished observations). However, in vivo, we did not find any evidence for direct infection of
CD452 nonhematopoietic cells or specifically of lung epithelial
cells (Fig. 2D; N. Joller and A. Oxenius, unpublished observations). Rather, GFP+ cells in the lung of mice infected with
a rGFP-expressing L. pneumophila strain were exclusively CD45+
F4/80+ CD11c+ AMs at early time points postinfection (Fig. 2D).
One day postinfection, infiltrating neutrophils and monocytes also
turned GFP+, but we consistently failed to detect L. pneumophila
in nonhematopoietic CD452 cells in the lung (Fig. 2D and data
not shown). The discrepancy between our in vitro and in vivo
findings highlights the complexity of the intact lung and emphasizes the relevance to study host–pathogen interactions in vivo
where the integrity of the host tissue is retained.
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CROSS TALK BETWEEN LUNG PARENCHYMA AND IMMUNE CELLS
The relevance of IL-1R signaling for chemokine production by
nonhematopoietic cells was further underlined by the finding that
chimeric mice in which nonhematopoietic cells selectively lacked
the IL-1R showed a strong defect in chemokine production at early
time points after L. pneumophila infection (Fig. 4A). This condition resulted in a strong decrease in neutrophil recruitment (Fig.
4B) and impaired control of L. pneumophila (Fig. 4C). Furthermore, comparable impairment of bacterial control was observed in
L. pneumophila-infected IL-1R2/2 mice (Fig. 4C).
We finally assessed which cells produced the IL-1b that acted on
nonhematopoietic cells in the airways of L. pneumophila-infected
mice. Whereas caspase-1 (which is involved in IL-1b processing)
was necessary for L. pneumophila-induced inflammation (Fig. 5A,
5B), it was not required in nonhematopoietic cells because chimeric mice lacking caspase-1 selectively in nonhematopoietic
cells showed no defect in chemokine production and leukocyte
infiltration into the airways (Fig. 5C, 5D). Furthermore, chimeric
mice with MyD88 deficiency in nonhematopoietic cells produced
normal levels of IL-1b in response to L. pneumophila infection
(Fig. 5E). These findings suggested that the hematopoietic compartment was the source of IL-1b. Indeed, we detected IL-1b in
AMs from L. pneumophila-infected mice (Fig. 5F). In addition,
IL-1b production was dependent on the expression of MyD88 in
hematopoietic cells (Fig. 5E). This was further corroborated by
the fact that MyD88-deficient macrophages were unable to express IL-1b protein, as shown previously (12), and mRNA in response to L. pneumophila infection in vitro (Fig. 5G, 5H). This
FIGURE 4. IL-1R signaling induces chemokine production in nonhematopoietic cells in vivo. A–C, Irradiation chimeras were generated by
reconstituting C57BL/6 or il-1r2/2 mice with C57BL/6 bone marrow.
Chemokine levels (A) and neutrophil counts (B) were determined in the
BAL of infected chimeras 4 h postinfection with L. pneumophila JR32. C,
Bacterial counts in the BAL were determined at 4 and 24 h after L.
pneumophila infection in B6 . B6 and B6 . il-1r2/2 chimeric mice (left
panel), or in nonchimeric il-1r2/2 or B6 mice (right panel). Data are mean
and SD of four mice per group (one uninfected mouse in A and B) and are
representative of two independent experiments each. *p , 0.05, **p ,
0.01, ***p , 0.001 (two-tailed unpaired t test).
defect in chemokine production was corroborated in vitro when
imAECs were stimulated with supernatants from L. pneumophilainfected, MyD88-deficient BMDM (Supplemental Fig. 4). Finally,
and consistent with the notion that L. pneumophila activates
caspase-1 in a T4SS-dependent manner (26–28), we found that IL1b production in the airways was abolished in mice infected with
a T4SS-deficient strain of L. pneumophila (Fig. 5I).
Discussion
Airway infections with pathogenic bacteria need to be rapidly
detected and controlled to secure a high degree of organ function,
which is essential for sufficient gas exchange. In this article, we show
that the swift induction of an inflammatory response including the
fast recruitment of infection-controlling neutrophils upon airway infection with the Gram-negative bacterium L. pneumophila critically
depends on both hematopoietic and nonhematopoietic cells in the
lung. Most importantly, the nonhematopoietic cells in the lung are
not merely a physical barrier for the lung tissue, but take an active
role in the buildup of the L. pneumophila-induced inflammatory
response. We demonstrate that this cross talk between lung AMs
and nonhematopoietic cells is chiefly regulated via IL-1b. Infection
of alveolar macrophages by L. pneumophila leads to the secretion
of IL-1b into the BAL, which is dependent on MyD88 on the host
side and on a functional T4SS on the pathogen side. IL-1b then
engages IL-1Rs on nonhematopoietic cells, including AECs, which
leads to the rapid induction of chemokine synthesis, with the neutrophil recruiting chemokines CXCL1 and CXCL2 being secreted
as early as 4 h postinfection. Thus, the dependence of the L.
pneumophila-induced inflammatory response on MyD88 is 2-fold:
first, MyD88 is essential for IL-1b induction in hematopoietic
cells; and second, nonhematopoietic cells depend on MyD88 for
IL-1R signaling. This cross talk between hematopoietic and non-
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FIGURE 3. IL-1R signaling induces chemokine production in nonhematopoietic cells in vitro. A, Wild type imAECs were stimulated with
the indicated doses of IL-1b, and chemokine induction was measured by
ELISA in the supernatant after 6 h. Data shown are mean and SD of
triplicate wells and representative of three independent experiments. B,
Schematic overview of the setup of in vitro cross-talk experiments with
BMDM and imAECs. C, Supernatant from L. pneumophila JR32-infected
BMDM was used to stimulate wild type and myd882/2 imAECs in presence or absence of IL-1Ra, as indicated, and chemokine induction in the
imAEC supernatant was measured by ELISA. Data shown are mean and
SD of triplicate wells and representative of three independent experiments.
D, The BAL from C57BL/6 mice infected with L. pneumophila JR32 for
2 h was used to stimulate wild type and myd882/2 imAECs, and chemokine induction in presence or absence of IL-1Ra was determined. Data
shown are mean and SD of triplicate wells and are representative of two
independent experiments. *p , 0.05, **p , 0.01, ***p , 0.001 (twotailed unpaired t test).
The Journal of Immunology
3135
hematopoietic cells expedites the early recruitment of neutrophils,
which, in turn, are crucial effectors in controlling airway L. pneumophila infection.
In this study, we have focused on analyzing the very early stages
of airway L. pneumophila infection where we have dissected an
important role of nonhematopoietic cells in orchestrating rapid
recruitment of PMNs. However, it is conceivable that other
mechanisms may compensate for an involvement of nonhematopoietic cells for PMN recruitment at later stages of infection. For
instance, bacterial replication, and hence increased PAMP levels,
might lead to more pronounced chemokine production by AMs
(24). Chemokine levels in the BAL were severely reduced at 4 h
after L. pneumophila infection in the absence of the IL-1b communication loop between AM and nonhematopoietic cells, but
they were still increased compared with noninfected control mice.
These low levels of CXCL1 and CXCL2 might be sufficient to
trigger PMN recruitment to some extent (Figs. 1B, 2C, 4B), and at
later time points, chemokine levels might further increase and thus
enhance PMN recruitment without involvement of nonhematopoietic cells. In addition, alternative PMN-attracting factors
such as the LPS-induced CXC chemokine (LIX) (29), lipids (30),
complement fragments (31), or IL-17 (32–34) might operate independently of nonhematopoietic cells in the lung.
Nonhematopoietic cells in the lung, in particular, epithelial cells,
have been shown, mostly in vitro using epithelial cell lines, to
directly possess pathogen-sensing capacities via membrane-bound
and cytosolic PRRs, and to translate such recognition events into
production and secretion of cytokines and chemokines, upregulation of cell surface adhesion molecules, and increased production
of antimicrobial peptides such as defensins and cathelicidins
(reviewed in Refs. 1, 4). As has been documented for intestinal
epithelium (35, 36), PAMP recognition by lung epithelial cells may
be more limited compared with innate immune cells to avoid recurrent inflammatory processes. This limitation may partly relate to
restricted PRR expression on epithelial surfaces (37), reduced expression of cosignaling molecules, or increased expression of inhibitory molecules (reviewed in Ref. 4). Nevertheless, the activated
lung epithelium closely communicates with other cellular players
of the innate immune system including phagocytes (neutrophils,
macrophages, and DCs), NK cells, and others (38–40).
In vivo, the exact contribution of parenchymal versus hematopoietic cells to the initiation and control of the immune response
within the lung remains elusive and seems to depend on the nature
of the inflammatory stimulus (i.e., type of pathogen, isolated PRR
agonist) as reported in various studies using chimeric mouse models. When the PRR agonist flagellin was administered intranasally,
nonhematopoietic cells control early chemokine production and
PMN infiltration via TLR5 signaling (41, 42). In contrast, inhalation
of endotoxin induced a cytokine response that depended on
MyD88 signaling in hematopoietic cells, whereas bronchoconstriction depended on MyD88 signaling on nonhematopoietic cells
(43), and in a study using TLR4-deficient chimeric mice, expression of TLR4 on hematopoietic cells and macrophages was crucial
to recruit neutrophils to the airspace after LPS treatment (44).
Downloaded from http://www.jimmunol.org/ by guest on July 31, 2017
FIGURE 5. IL-1b is produced by AMs in an MyD88- and T4SS-dependent manner. A and B, Chemokine levels (A) and neutrophil recruitment (B) were
measured in the BAL of caspase-12/2 and C57BL/6 control mice 4 h postinfection with L. pneumophila JR32. Data are mean and SD of three mice per
group and are representative of two independent experiments. C and D, Irradiation chimeras were generated by reconstituting C57BL/6 or caspase-12/2
mice with C57BL/6 bone marrow. Chemokine levels (C) and neutrophil counts (D) were determined in the BAL of infected chimeras 4 h postinfection
with L. pneumophila JR32. Data are mean and SD of five mice per group pooled from two independent experiments. E, C57BL/6 mice and chimeras
wherein either all nonhematopoietic cells (B6 . MyD882/2) or all hematopoietic cells (MyD882/2 . B6) were MyD88-deficient were infected with L.
pneumophila JR32, and IL-1b secretion in the BAL was determined 4 h postinfection. Data are mean and SD of two mice per group and are representative
of three independent experiments. F, CD11c+ cells were purified from the BAL of mice infected 3 h previously with JR32 or DT or from uninfected
control mice, and pro–IL-1b was analyzed by Western blotting. G and H, Wild type and MyD88-deficient BMDM were infected with the indicated
multiplicity of infections of L. pneumophila JR32, and secreted IL-1b protein (G) or IL-1b mRNA (H) was quantified. Data are mean and SD of triplicate
measurements. I, IL-1b was measured in the BAL of C57BL/6 mice infected with L. pneumophila strain JR32 or DT for 4 h. Data are mean and SD of
four infected mice per group and two control mice, and are representative of three independent experiments. *p , 0.05, **p , 0.01, ***p , 0.001 (twotailed unpaired t test).
3136
CROSS TALK BETWEEN LUNG PARENCHYMA AND IMMUNE CELLS
Acknowledgments
We thank W.-D. Hardt for various mouse strains, C. Halin for the antipodoplanin Ab, T. Kündig for the IL-1Ra, the staff of our animal facility
for the care of the animals used in this study, and members of the Oxenius
group for discussions.
Disclosures
The authors have no financial conflicts of interest.
References
1. Bartlett, J. A., A. J. Fischer, and P. B. McCray, Jr. 2008. Innate immune functions
of the airway epithelium. Contrib. Microbiol. 15: 147–163.
2. Lambrecht, B. N., and H. Hammad. 2009. Biology of lung dendritic cells at the
origin of asthma. Immunity 31: 412–424.
3. Diamond, G., D. Legarda, and L. K. Ryan. 2000. The innate immune response of
the respiratory epithelium. Immunol. Rev. 173: 27–38.
4. Hippenstiel, S., B. Opitz, B. Schmeck, and N. Suttorp. 2006. Lung epithelium as
a sentinel and effector system in pneumonia—molecular mechanisms of pathogen recognition and signal transduction. Respir. Res. 7: 97.
5. Tateda, K., T. A. Moore, M. W. Newstead, W. C. Tsai, X. Zeng, J. C. Deng,
G. Chen, R. Reddy, K. Yamaguchi, and T. J. Standiford. 2001. Chemokinedependent neutrophil recruitment in a murine model of Legionella pneumonia:
potential role of neutrophils as immunoregulatory cells. Infect. Immun. 69:
2017–2024.
6. Archer, K. A., and C. R. Roy. 2006. MyD88-dependent responses involving tolllike receptor 2 are important for protection and clearance of Legionella pneu-
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
mophila in a mouse model of Legionnaires’ disease. Infect. Immun. 74: 3325–
3333.
Archer, K. A., L. Alexopoulou, R. A. Flavell, and C. R. Roy. 2009. Multiple
MyD88-dependent responses contribute to pulmonary clearance of Legionella
pneumophila. Cell. Microbiol. 11: 21–36.
Spörri, R., N. Joller, U. Albers, H. Hilbi, and A. Oxenius. 2006. MyD88dependent IFN-g production by NK cells is key for control of Legionella
pneumophila infection. J. Immunol. 176: 6162–6171.
Hawn, T. R., K. D. Smith, A. Aderem, and S. J. Skerrett. 2006. Myeloid differentiation primary response gene (88)- and toll-like receptor 2-deficient mice
are susceptible to infection with aerosolized Legionella pneumophila. J. Infect.
Dis. 193: 1693–1702.
Isberg, R. R., T. J. O’Connor, and M. Heidtman. 2009. The Legionella pneumophila replication vacuole: making a cosy niche inside host cells. Nat. Rev.
Microbiol. 7: 13–24.
Coers, J., C. Monahan, and C. R. Roy. 1999. Modulation of phagosome biogenesis by Legionella pneumophila creates an organelle permissive for intracellular growth. Nat. Cell Biol. 1: 451–453.
Shin, S., C. L. Case, K. A. Archer, C. V. Nogueira, K. S. Kobayashi,
R. A. Flavell, C. R. Roy, and D. S. Zamboni. 2008. Type IV secretion-dependent
activation of host MAP kinases induces an increased proinflammatory cytokine
response to Legionella pneumophila. PLoS Pathog. 4: e1000220.
Akamine, M., F. Higa, N. Arakaki, K. Kawakami, K. Takeda, S. Akira, and
A. Saito. 2005. Differential roles of Toll-like receptors 2 and 4 in in vitro responses
of macrophages to Legionella pneumophila. Infect. Immun. 73: 352–361.
Archer, K. A., F. Ader, K. S. Kobayashi, R. A. Flavell, and C. R. Roy. 2010.
Cooperation between multiple microbial pattern recognition systems is important for host protection against the intracellular pathogen Legionella pneumophila. Infect. Immun. 78: 2477–2487.
Adachi, O., T. Kawai, K. Takeda, M. Matsumoto, H. Tsutsui, M. Sakagami,
K. Nakanishi, and S. Akira. 1998. Targeted disruption of the MyD88 gene results
in loss of IL-1- and IL-18-mediated function. Immunity 9: 143–150.
Labow, M., D. Shuster, M. Zetterstrom, P. Nunes, R. Terry, E. B. Cullinan,
T. Bartfai, C. Solorzano, L. L. Moldawer, R. Chizzonite, and K. W. McIntyre.
1997. Absence of IL-1 signaling and reduced inflammatory response in IL-1 type
I receptor-deficient mice. J. Immunol. 159: 2452–2461.
Li, P., H. Allen, S. Banerjee, S. Franklin, L. Herzog, C. Johnston, J. McDowell,
M. Paskind, L. Rodman, J. Salfeld, et al. 1995. Mice deficient in IL-1 betaconverting enzyme are defective in production of mature IL-1 beta and resistant to endotoxic shock. Cell 80: 401–411.
Jat, P. S., M. D. Noble, P. Ataliotis, Y. Tanaka, N. Yannoutsos, L. Larsen, and
D. Kioussis. 1991. Direct derivation of conditionally immortal cell lines from an
H-2Kb-tsA58 transgenic mouse. Proc. Natl. Acad. Sci. USA 88: 5096–5100.
Sadosky, A. B., L. A. Wiater, and H. A. Shuman. 1993. Identification of
Legionella pneumophila genes required for growth within and killing of human
macrophages. Infect. Immun. 61: 5361–5373.
Tiaden, A., T. Spirig, S. S. Weber, H. Brüggemann, R. Bosshard, C. Buchrieser,
and H. Hilbi. 2007. The Legionella pneumophila response regulator LqsR promotes host cell interactions as an element of the virulence regulatory network
controlled by RpoS and LetA. Cell. Microbiol. 9: 2903–2920.
Segal, G., M. Purcell, and H. A. Shuman. 1998. Host cell killing and bacterial
conjugation require overlapping sets of genes within a 22-kb region of the
Legionella pneumophila genome. Proc. Natl. Acad. Sci. USA 95: 1669–1674.
Schmeck, B., P. D. N’Guessan, M. Ollomang, J. Lorenz, J. Zahlten, B. Opitz,
A. Flieger, N. Suttorp, and S. Hippenstiel. 2007. Legionella pneumophila-induced NF-kappaB- and MAPK-dependent cytokine release by lung epithelial
cells. Eur. Respir. J. 29: 25–33.
Fuse, E. T., K. Tateda, Y. Kikuchi, T. Matsumoto, F. Gondaira, A. Azuma,
S. Kudoh, T. J. Standiford, and K. Yamaguchi. 2007. Role of Toll-like receptor 2
in recognition of Legionella pneumophila in a murine pneumonia model. J. Med.
Microbiol. 56: 305–312.
De Filippo, K., R. B. Henderson, M. Laschinger, and N. Hogg. 2008. Neutrophil
chemokines KC and macrophage-inflammatory protein-2 are newly synthesized
by tissue macrophages using distinct TLR signaling pathways. J. Immunol. 180:
4308–4315.
Shahangian, A., E. K. Chow, X. Tian, J. R. Kang, A. Ghaffari, S. Y. Liu,
J. A. Belperio, G. Cheng, and J. C. Deng. 2009. Type I IFNs mediate development of postinfluenza bacterial pneumonia in mice. J. Clin. Invest. 119:
1910–1920.
Molofsky, A. B., B. G. Byrne, N. N. Whitfield, C. A. Madigan, E. T. Fuse,
K. Tateda, and M. S. Swanson. 2006. Cytosolic recognition of flagellin by mouse
macrophages restricts Legionella pneumophila infection. J. Exp. Med. 203:
1093–1104.
Zamboni, D. S., K. S. Kobayashi, T. Kohlsdorf, Y. Ogura, E. M. Long,
R. E. Vance, K. Kuida, S. Mariathasan, V. M. Dixit, R. A. Flavell, et al. 2006.
The Birc1e cytosolic pattern-recognition receptor contributes to the detection
and control of Legionella pneumophila infection. Nat. Immunol. 7: 318–325.
Ren, T., D. S. Zamboni, C. R. Roy, W. F. Dietrich, and R. E. Vance. 2006.
Flagellin-deficient Legionella mutants evade caspase-1- and Naip5-mediated
macrophage immunity. PLoS Pathog. 2: e18.
Rovai, L. E., H. R. Herschman, and J. B. Smith. 1998. The murine neutrophilchemoattractant chemokines LIX, KC, and MIP-2 have distinct induction kinetics, tissue distributions, and tissue-specific sensitivities to glucocorticoid
regulation in endotoxemia. J. Leukoc. Biol. 64: 494–502.
Ford-Hutchinson, A. W., M. A. Bray, M. V. Doig, M. E. Shipley, and
M. J. Smith. 1980. Leukotriene B, a potent chemokinetic and aggregating substance released from polymorphonuclear leukocytes. Nature 286: 264–265.
Downloaded from http://www.jimmunol.org/ by guest on July 31, 2017
Also in the context of bacterial airway infections, nonhematopoietic cells in the lung were previously attributed a role in
innate immune defense: pneumonia induced by infection with
Klebsiella pneumoniae is controlled by nonhematopoietic cells by
directly ingesting and degrading bacteria, and by facilitating their
opsonization (45). Furthermore, nonhematopoietic cells in the
lung were shown to contribute to cytokine production upon exposure to Bacillus anthracis spores (46) or Haemophilus influenzae infection (47). Finally, in P. aeruginosa-infected mouse lungs,
MyD88 in nonhematopoietic cells is essential for early control of
infection, whereas at later phases of the infection, MyD88 expression in both parenchymal and hematopoietic cells contributes
to control pathogen replication (48).
In this study, we dissected the cross talk between nonhematopoietic and hematopoietic cells during early airway L. pneumophila
infection-induced inflammation. This cellular interplay does not rely
on direct sensing of PAMPs by nonhematopoietic cells themselves,
but rather positions them as potent amplifiers between L. pneumophila-infected AMs and PMN recruitment, the latter being essential
for early reduction in pathogen load.
Of interest, a similar cross talk between nonhematopoietic and
hematopoietic cells has been described in a murine cutaneous
infection model with Staphylococcus aureus, where MyD88- and
IL-1R–deficient mice showed impaired neutrophil recruitment to
the site of infection. This process is independent of IL-1R/MyD88
in hematopoietic cells, suggesting that IL-1R/MyD88 signaling in
skin-resident nonhematopoietic cells is critical to promote neutrophil recruitment (49).
Taken together, our results contribute to the understanding of the
early events that control the initiation of the inflammatory response
upon L. pneumophila airway infection. Nonhematopoietic cells,
including AECs, which form a tight boundary and line the entire
airspace of the lung, thus perform many more functions than just
that of a physical barrier preventing pathogens from invading the
underlying tissues. Our data add an important example to the recently recognized role of nonhematopoietic cells in the lung (but
also in other organs such as the gut and the skin) to secrete specific
inflammatory mediators, which can have various functions but
often promote key decisions in innate and adaptive immune cells
(50).
The Journal of Immunology
42.
43.
44.
45.
46.
47.
48.
49.
50.
expressing TLR5 control the respiratory epithelium’s innate immune responses
to flagellin. Eur. J. Immunol. 39: 1587–1596.
Feuillet, V., S. Medjane, I. Mondor, O. Demaria, P. P. Pagni, J. E. Galán,
R. A. Flavell, and L. Alexopoulou. 2006. Involvement of Toll-like receptor 5 in
the recognition of flagellated bacteria. Proc. Natl. Acad. Sci. USA 103: 12487–
12492.
Noulin, N., V. F. Quesniaux, S. Schnyder-Candrian, B. Schnyder, I. Maillet,
T. Robert, B. B. Vargaftig, B. Ryffel, and I. Couillin. 2005. Both hemopoietic
and resident cells are required for MyD88-dependent pulmonary inflammatory
response to inhaled endotoxin. J. Immunol. 175: 6861–6869.
Hollingsworth, J. W., B. J. Chen, D. M. Brass, K. Berman, M. D. Gunn,
D. N. Cook, and D. A. Schwartz. 2005. The critical role of hematopoietic cells in
lipopolysaccharide-induced airway inflammation. Am. J. Respir. Crit. Care Med.
171: 806–813.
Cortés, G., D. Alvarez, C. Saus, and S. Albertı́. 2002. Role of lung epithelial
cells in defense against Klebsiella pneumoniae pneumonia. Infect. Immun. 70:
1075–1080.
Chakrabarty, K., W. Wu, J. L. Booth, E. S. Duggan, N. N. Nagle,
K. M. Coggeshall, and J. P. Metcalf. 2007. Human lung innate immune response
to Bacillus anthracis spore infection. Infect. Immun. 75: 3729–3738.
Wang, X., C. Moser, J. P. Louboutin, E. S. Lysenko, D. J. Weiner, J. N. Weiser,
and J. M. Wilson. 2002. Toll-like receptor 4 mediates innate immune responses
to Haemophilus influenzae infection in mouse lung. J. Immunol. 168: 810–815.
Hajjar, A. M., H. Harowicz, H. D. Liggitt, P. J. Fink, C. B. Wilson, and
S. J. Skerrett. 2005. An essential role for non-bone marrow-derived cells in
control of Pseudomonas aeruginosa pneumonia. Am. J. Respir. Cell Mol. Biol.
33: 470–475.
Miller, L. S., R. M. O’Connell, M. A. Gutierrez, E. M. Pietras, A. Shahangian,
C. E. Gross, A. Thirumala, A. L. Cheung, G. Cheng, and R. L. Modlin. 2006.
MyD88 mediates neutrophil recruitment initiated by IL-1R but not TLR2 activation in immunity against Staphylococcus aureus. Immunity 24: 79–91.
Swamy, M., C. Jamora, W. Havran, and A. Hayday. 2010. Epithelial decision
makers: in search of the ‘epimmunome’. Nat. Immunol. 11: 656–665.
Downloaded from http://www.jimmunol.org/ by guest on July 31, 2017
31. Lee, J., G. Cacalano, T. Camerato, K. Toy, M. W. Moore, and W. I. Wood. 1995.
Chemokine binding and activities mediated by the mouse IL-8 receptor. J.
Immunol. 155: 2158–2164.
32. Zhang, X., L. Gao, L. Lei, Y. Zhong, P. Dube, M. T. Berton, B. Arulanandam,
J. Zhang, and G. Zhong. 2009. A MyD88-dependent early IL-17 production
protects mice against airway infection with the obligate intracellular pathogen
Chlamydia muridarum. J. Immunol. 183: 1291–1300.
33. Ye, P., F. H. Rodriguez, S. Kanaly, K. L. Stocking, J. Schurr, P. Schwarzenberger,
P. Oliver, W. Huang, P. Zhang, J. Zhang, et al. 2001. Requirement of interleukin
17 receptor signaling for lung CXC chemokine and granulocyte colonystimulating factor expression, neutrophil recruitment, and host defense. J. Exp.
Med. 194: 519–527.
34. Ferretti, S., O. Bonneau, G. R. Dubois, C. E. Jones, and A. Trifilieff. 2003. IL-17,
produced by lymphocytes and neutrophils, is necessary for lipopolysaccharideinduced airway neutrophilia: IL-15 as a possible trigger. J. Immunol. 170: 2106–
2112.
35. Abreu, M. T., M. Fukata, and M. Arditi. 2005. TLR signaling in the gut in health
and disease. J. Immunol. 174: 4453–4460.
36. Cario, E., and D. K. Podolsky. 2005. Intestinal epithelial TOLLerance versus
inTOLLerance of commensals. Mol. Immunol. 42: 887–893.
37. Guillot, L., S. Medjane, K. Le-Barillec, V. Balloy, C. Danel, M. Chignard, and
M. Si-Tahar. 2004. Response of human pulmonary epithelial cells to lipopolysaccharide involves Toll-like receptor 4 (TLR4)-dependent signaling pathways:
evidence for an intracellular compartmentalization of TLR4. J. Biol. Chem. 279:
2712–2718.
38. Lambrecht, B. N., J. B. Prins, and H. C. Hoogsteden. 2001. Lung dendritic cells
and host immunity to infection. Eur. Respir. J. 18: 692–704.
39. Delclaux, C., and E. Azoulay. 2003. Inflammatory response to infectious pulmonary injury. Eur. Respir. J. Suppl. 42: 10s–14s.
40. Nicod, L. P. 1999. Pulmonary defence mechanisms. Respiration 66: 2–11.
41. Janot, L., J. C. Sirard, T. Secher, N. Noulin, L. Fick, S. Akira, S. Uematsu,
A. Didierlaurent, T. Hussell, B. Ryffel, and F. Erard. 2009. Radioresistant cells
3137