Neutrophil-Toxin Interactions Promote Antigen Delivery and
Mucosal Clearance of Streptococcus pneumoniae1
Kathryn A. Matthias,* Aoife M. Roche,* Alistair J. Standish,* Mikhail Shchepetov,*
and Jeffrey N. Weiser2*†
Delivery of Ag to inductive sites, such as nasal-associated lymphoid tissue (NALT) or GALT, is thought to promote mucosal
immunity. Host and microbial factors that contribute to this process were investigated during model murine airway colonization
by the pathogen Streptococcus pneumoniae. Colonization led to the deposition of released bacterial capsular Ag in the NALT in
a manner consistent with trafficking through M cells. This Ag was derived from processing of bacteria in the lumen of the
paranasal spaces rather than through invasion or sampling of intact bacteria. Neutrophils, which are recruited to the paranasal
spaces where they associate with and may degrade bacteria, were required for efficient Ag delivery. Maximal Ag delivery to the
NALT also required expression of the bacterial toxin pneumolysin. Pneumolysin and pneumolysin-expressing bacteria lysed
neutrophils through pore formation in vitro. Accordingly, a pneumolysin-dependent loss of neutrophils, which correlated with the
increased release of bacterial products, was observed in vivo. Thus, delivery of Ag to the NALT was enhanced by neutrophil-
mediated generation of bacterial products together with bacterial-induced lysis of neutrophils. The impaired Ag delivery of pneumo-
lysin-deficient bacteria was associated with diminished clearance from the mucosal surface. This study demonstrates how microbial-host
interactions affect Ag delivery and the effectiveness of mucosal immunity. The Journal of Immunology, 2008, 180: 6246–6254.
recognition of microbial products stimulating an immune response
that leads to eventual clearance. Microbial and host factors that
affect inflammation and the development of mucosal immunity
during colonization remain poorly understood. Successful mucosal
pathogens often express virulence factors such as toxins that in-
hibit the antimicrobial activity of recruited inflammatory cells (1).
Microbial manipulation of these innate immune responses may, in
turn, affect the generation of adaptive immunity, altering the dy-
namics of colonization and infection (2). This report focuses spe-
cifically on how these host-microbe interactions may affect the
delivery of Ag to the mucosal immune system.
In a prior study of experimental human colonization with Strep-
tococcus pneumoniae (the pneumococcus), the duration of bacte-
rial carriage of a type 23F isolate ranged from 3 to 122 days (3).
When mice were challenged intranasally with the same isolate and
inoculum, the duration of colonization and the resulting immune
response were similar to those of the natural human host (4). Colo-
nization of both humans and mice resulted in the production of cap-
sular polysaccharide-specific Abs, which was associated with reduced
carriage when provided as an immunogen (5, 6). The loss of coloni-
zation, however, was shown to require cellular immunity in animal
models, because MHC class II-deficient mice lacking CD4?T cells
olonization of mucosal surfaces is the initial interaction
with a host for many commensal and pathogenic mi-
crobes. However, this interaction may be short-lived with
polysaccharide is generally considered a T cell-independent Ag.
In the human upper respiratory tract, the tonsils and adenoids
constitute a primary site of exposure to inhaled pathogens. The
Waldeyer’s ring, as the tissues are collectively called, contains T
and B cells and is thought to be important in adaptive immune re-
sponses to respiratory flora, including S. pneumoniae (9, 10). Mice
lack the Waldeyer’s ring but contain the analogous nasal-associated
lymphoid tissue (NALT),3which extends the length of the ventral
surface of the upper palate and has been implicated as an inductive
site for specific T cell priming of streptococcal Ags (11–13). Park et
al. proposed that T cell priming was initiated when Streptococcus
pyogenes directly entered the NALT through M cells on the adjacent
mucosal surface (14). If the NALT acts similarly as an inductive site
upon pneumococcal infection, then delivery of pneumococcal Ag to
the NALT should enhance bacterial clearance.
We previously demonstrated that pneumococcal colonization of
the murine upper respiratory tract led to an acute inflammatory
response characterized by the influx of neutrophils into the para-
nasal spaces (7). These neutrophils, which were observed for ?7
days postinoculation, appeared to be insufficient to control initial
mucosal colonization but may affect the immune response and con-
tribute to the pathogen’s eventual clearance (15). An important
determinant of the inflammatory response to the pneumococcus is
its sole toxin, pneumolysin, a member of a family of thiol-
activated, cholesterol-binding cytotoxins expressed by many
Gram-positive pathogens. Pneumolysin has been associated
with multiple effects including the stimulation of neutrophil re-
cruitment and T cell responses (7, 16–18).
*Department of Microbiology and†Department of Pediatrics, University of Pennsyl-
vania School of Medicine, Philadelphia, PA 19104
Received for publication November 2, 2007. Accepted for publication February 29, 2008.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1This work was supported by grants from the U.S. Public Health Service to J.N.W.
(AI44231 and AI38446) and the Morphology Core of the Center for the Molecular
Studies of Liver and Digestive Disease (Center Grant P30 DK50306).
2Address correspondence and reprint requests to Dr. Jeffrey N. Weiser, 402A John-
son Pavilion, Department of Microbiology and Pediatrics, University of Pennsylvania,
Philadelphia, PA 19104. E-mail address: email@example.com
3Abbreviations used in this paper: NALT, nasal-associated lymphoid tissue; IF, im-
munofluorescence; DAPI, 4?,6-diamidino-2-phenylindole; Ply, purified pneumolysin;
PdB, purified pneumolysin toxoid; LDH, lactate dehydrogenase; wt, wild type;
UEA-1, Ulex europaeus agglutinin-1.
Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00
The Journal of Immunology
In this report, we illustrate that the interaction of host neutro-
phils with the pore-forming toxin pneumolysin leads to release and
delivery of pneumococcal Ag to the NALT and correlates with
more rapid clearance of S. pneumoniae from the nasopharynx.
These results demonstrate the importance of the acute inflamma-
tory response in pneumococcal clearance, not as a direct means of
controlling infection, but rather as an enhancer of adaptive
Materials and Methods
Bacterial strains and culture
S. pneumoniae strains were grown in tryptic soy medium as described
elsewhere (19). Strain P1121, a type 23F isolate obtained from a study of
experimental human colonization, was selected for its ability to efficiently
colonize the murine nasopharynx (3, 4). The construction of defined mu-
tants of P1121 using the bicistronic positively and negatively selectable
Janus cassette technology was previously described and included: 1) ply?,
a strain containing an unmarked, complete in-frame deletion of the pneu-
molysin gene, and 2) Ply-W433F, a strain with the deletion restored with a
point mutation previously shown to reduce the efficiency of pore formation
95–99% (20–23). The genotype of ply?and Ply-W433F was confirmed by
sequencing across the introduced mutation. Expression of pneumolysin
was assessed by Western blot analysis probing with anti-pneumolysin Ab
(Novocastra Laboratories). A horse erythrocyte lysis assay was used to
assess pore formation as previously described (24). An encapsulated type
4 isolate (TIGR4) was also used in immunofluorescence (IF) (3) studies on
NALT. All strains were passaged intranasally in mice before preparation of
Animal colonization model
Female BALB/c mice (5–8 wk of age) were obtained from Taconic Lab-
oratories and housed in accordance with the protocols established by the
Institutional Animal Care and Use Committee. Mice were colonized for a
period of 1–25 days as previously described (4, 25). Briefly, an intranasal
inoculation of 107CFU (3) of PBS-washed, mid-log phase bacteria in 10
?l was administered drop-wise to the external nares. This procedure was
performed without anesthesia to minimize the possibility of aspiration into
the lungs. Following inoculation, at the specified time point, mice were
sacrificed and the tracheas cannulated. A total volume of 200 ?l of PBS
was instilled into the trachea and recovered from the nares for quantitative
culture. Serial dilutions of upper respiratory tract lavage were plated on
tryptic soy medium with catalase and neomycin (20 ?g/ml) to minimize the
growth of contaminants. The ply?and Ply-W433F mutants were plated on
selective medium with streptomycin (100 ?g/ml). The lower limit of de-
tection for bacteria obtained from the nasal lavage was 20 CFU/ml.
RB6-8C5, a rat anti-mouse IgG2b mAb directed against Ly-6G on the
surface of murine myeloid (and limited subpopulations of lymphoid) lin-
eage cells, was purified from ascites of nude mice given the RB6-8C5
hybridoma (26, 27). Neutrophil depletion was achieved by i.p. delivery of
100 ?g mAb per animal 24 h before intranasal inoculation. This treatment
was shown in pilot experiments to result in peripheral blood neutropenia
(?50 granulocytes/?l) for a period of at least 4 days. Control animals were
administered the equivalent dose of total rat IgG (Sigma-Aldrich).
The nasal lavages of five mice from each group were pooled, centrifuged
at 1500 ? g, and resuspended in 1% BSA in PBS. Nonspecific binding was
blocked using a rat anti-mouse mAb directed against the Fc?III/II receptor
(CD16/CD32) and the following Abs were applied: rat anti-mouse Ly-6G
to detect neutrophils; rat anti-mouse CD11b, a cell-surface marker of neu-
trophil activation; hamster anti-mouse CD11c to detect dendritic cells; rat
anti-mouse CD14 to detect monocytes; and rat anti-mouse F4/80 to detect
macrophages. All Abs were obtained from BD Biosciences except for anti-
F4/80, which was purchased from eBioscience. A total of 10,000 cells were
collected for each sample and groups were compared using FlowJo soft-
ware (Tree Star).
Histology and IF
Three days postinoculation, mice were sacrificed and decapitated. Heads
were treated as described elsewhere (15). Briefly, samples were fixed for
48 h in formalin (4% paraformaldehyde) and decalcified for 30 days in 0.12
M EDTA (pH 7.0). The heads were then frozen in Tissue-Tek OCT em-
bedding medium (Electron Microscopy Sciences) and 5-?m sections cut,
and either stained with H&E or stored at ?80°C for IF.
For IF labeling of bacteria, neutrophils, macrophages, and dendritic
cells, all sections were postfixed in 1/1 methanol-acetone at ?20°C for 10
min followed by washing in PBS. Nonspecific binding was inhibited by
incubation (10 min) in protein-blocking reagent (Coulter-Immunotech).
Bacteria were detected using specific antisera (diluted 1/500) recognizing
the type 23F or type 4 capsular polysaccharide (Staten Serum Institut)
followed by Cy3-conjugated anti-rabbit Ig secondary Abs (diluted 1/400,
Jackson ImmunoResearch). Neutrophils were detected by application of
anti-Ly-6G (diluted 1/500) and Cy2-conjugated anti-rabbit Igs (diluted
1/400, Jackson ImmunoResearch). Dendritic cells and macrophages were
detected using the same technique as that for neutrophil detection, except
for use of appropriate primary and secondary Abs (Armenian hamster anti-
mouse CD11c and anti-hamster-Cy2 for dendritic cells; rat anti-mouse
F4/80 and anti-rat-Cy2 for macrophages). Murine cells were visualized by
counterstaining with 4?,6-diamidino-2-phenylindole (DAPI) (Molecular
The same procedure as above was used to selectively label M cells,
except postfixing was performed in acetone alone and nonspecific blocking
with protein-blocking reagent was followed by application of the Avidin/
Biotin Blocking Kit according to the manufacturer’s protocol (Vector Lab-
oratories). Bacteria were detected as described above, and sections were
colabeled with 50 ?g/ml of a biotinylated lectin (Ulex europaeus aggluti-
nin-1 (UEA-1), Vector Laboratories), which has been shown to selectively
label M cells in the gut and the nasal mucosa (14). UEA-1 labeling was
detected with a Cy2-conjugated anti-avidin secondary Ab (Jackson Immu-
noResearch) and cells were counterstained with DAPI.
All IF imaging was performed on a Nikon Eclipse E600 microscope
equipped with a high-resolution charge-coupled device digital camera
(CoolSNAP cf, Roper Scientific). Image analysis was conducted using
IPLAB software (Scanalytics).
Gentamicin protection assay
To determine whether viable pneumococci were present within cells, the
NALT was dissected from mice as described previously after 3 days of
infection with the wild-type (wt) strain (28, 29). Cells were passed through
a wire mesh screen to generate a single-cell suspension and incubated with
gentamicin sulfate (final concentration 300 ?g/ml) at 37°C for 2 h in RPMI
1640 (Invitrogen), conditions reported in a previous study to be sufficient
to kill extra- but not intracellular strain P1121 (30). The cells were spun
down and washed to remove the antibiotic, and serial dilution plating was
used to determine the number of viable intracellular bacteria.
Nasal lavage and serum samples were tested following 25 days of bacterial
infection for levels of anti-pneumococcal IgA or IgG and IgM Abs, re-
spectively. Whole-cell wt bacteria grown on overnight cultures of tryptic
soy medium were suspended in coating buffer (0.015 M Na2CO3, 0.035 M
NaHCO3(pH 9.6)) and used to coat 96-well Immulon 2HB plates (Thermo
Electron) at an OD of 0.1. Alternatively, plates were coated with purified
pneumococcal surface protein A (0.5 ?g/ml). Plates were incubated over-
night at 4°C, washed with 0.05% PBS-Brij, and blocked by 1 h incubation
in 1% BSA (Sigma-Aldrich). After blocking, samples were plated in 2-fold
serial dilutions and incubated overnight at 4°C. Samples were then washed
in PBS-Brij, and Ag-specific Abs were detected by 2 h incubation (37°C)
in alkaline phosphatase-conjugated goat anti-mouse Igs (anti-IgA for la-
vage samples, anti-IgG/anti-IgM for serum, at a dilution of 1/4000) (Sig-
ma-Aldrich). Plates were developed by 1 h incubation in p-nitrophenyl
phosphate (Sigma-Aldrich) and absorbencies were recorded at 415 nm.
Geometric mean titers were assessed by calculating the sample dilution at
which A ? 0.1.
MIP-2 levels were measured in nasal lavage specimens using a MIP-2
Quantikine ELISA kit (R&D Systems).
Isolation of neutrophils and phagocytic killing assay
Neutrophil-enriched peritoneal exudate cells were isolated from BALB/c
mice as previously described (31). Briefly, mice were administered two
separate 1.0 ml i.p. injections of 10% casein in PBS 24 h and 2 h before cell
harvest. Animals were sacrificed and phagocytes obtained by peritoneal
lavage using 8 ml of 0.1% gelatin in HBSS without Mg2?or Ca2?(In-
vitrogen). Phagocytes were enriched for neutrophils by separation in a
Ficoll density gradient centrifugation using Mono-Poly resolving medium
(MP Biomedicals). Cells were then collected and washed with 5 ml of
HBSS and counted by trypan blue exclusion.
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6254 ANTIGEN DELIVERY DURING S. pneumoniae COLONIZATION