Expression of capsular polysaccharide by bacterial pathogens is associated with increased resistance to host clearance mechanisms, in particular by evading opsonization and uptake by professional phagocytes. The potential for rapid progression of disease caused by encapsulated bacteria points to the importance of innate immunity at the mucosal surface where infection is initiated. Using a murine model of nasopharyngeal colonization, host immune components that contribute to the mucosal clearance of capsule-expressing bacteria were investigated. Clearance of encapsulated Haemophilus influenzae (Hi) required both TLR and nucleotide-binding oligomerization domain (NOD) signaling pathways, whereas individual deficiencies in each of these signaling cascades did not affect clearance of nonencapsulated strains. Moreover, clearance of Hi-expressing capsular polysaccharide required the recruitment of neutrophils to the site of infection, and ex vivo phagocytic bacterial killing required expression of the NOD1 signaling pathway. Conversely, redundancies within these innate immune pathways of non-neutrophil cells were sufficient to promote mucosal clearance of nonencapsulated Hi. Our findings reveal a role for NOD1 in protection from encapsulated pathogens. In addition, this study provides an example of a microbial virulence determinant that alters the requirements for host signaling to provide effective protection.

Many major bacterial pathogens express a thick coat of capsular polysaccharide on their surface. Their capsules render them more resistant to host clearance mechanisms, particularly those involving opsonization by complement and/or Ab followed by uptake by professional phagocytes (1, 2). These attributes enhance the ability of extracellular bacteria to survive within the bloodstream and explain why encapsulated pathogens are among the most common agents causing severe invasive infections. However, many encapsulated organisms reside on mucosal surfaces where they exist primarily in a commensal relationship with their host. In the human nasopharynx, clinically important examples of these encapsulated organisms include Haemophilus influenzae (Hi)3 type b (Hib), Streptococcus pneumoniae, and Neisseria meningitidis. Nasal carriage of these species is generally transient but a prerequisite for the development of disease (3, 4). Invasive infection is a relatively rare outcome but it may be both overwhelming and expedient, occurring within days from the establishment of colonization. Although capsular polysaccharides are often immunodominant Ags, adaptive immune responses may develop too slowly to confer protection during the initial period following bacterial acquisition. This suggests that innate immunity must be crucial for protection against encapsulated mucosal pathogens.

The cellular components of innate immunity use pattern recognition molecules (PRMs) that recognize microbial expressed pathogen-associated molecular patterns. Bacterial pathogen-associated molecular patterns trigger responses through interactions with PRMs including TLRs and nucleotide oligomerization domain (NOD) proteins (for review see Refs. 5, 6). In particular, TLR4 and TLR2 signaling pathways represent key surface regulators of innate immune responses to extracellular bacteria through the recognition of bacterial LPS and lipid-modified components, respectively. Additionally, the cytoplasmic NOD proteins are involved in innate immune signaling events through recognition of specific peptidoglycan motifs. NOD1, for instance, signals in response to a diaminopimelic acid-containing peptide moiety (7, 8). After recognition of bacterial components by these PRMs, distinct intracellular signaling events are elicited, resulting in the eventual activation of NFκB and initiation and/or modulation of innate and adaptive immune responses. Cooperation and redundancy among these innate immune detectors is of critical importance to regulating and shaping antimicrobial immunity.

Capsule-mediated modulation of bacterial interactions with PRMs has been suggested for a variety of bacterial species including S. pneumoniae (9), Salmonella enterica serotype typhi (10), and Streptococcus suis (11). Encapsulated organisms transiently residing within the nasopharynx are capable of causing rapid and serious infections as a result of overcoming host defense mechanisms. Therefore, we set forth to investigate the host immune components required for mucosal clearance of bacteria in vivo. In this study, Hi was used as a model pathogen since both capsule-expressing and nonexpressing (or nontypeable) forms naturally exist and are capable of initiating infection, and a role of capsule in facilitating bacterial survival within the host has been well established (12, 13). We found that in the absence of individual PRMs, clearance of nonencapsulated bacteria remains effective. Conversely, the absence of TLR2, TLR4, or NOD1 signaling pathways, and depletion of neutrophil-like cells, attenuates clearance of Hi-expressing capsular polysaccharide. Our findings highlight the differing requirements for protection by innate immune mechanisms for encapsulated and nonencapsulated mucosal pathogens.

The following strains of mice were obtained from Jackson ImmunoResearch Laboratories: C57BL6 (wild type (WT)), B6.129-S2-Igh-6tm1Cgn/J (μMT), B6.CB17-Prkdcscid/SzJ (SCID), and C57BL10ScNJ (TLR4−/−). μMT mice contain a targeted mutation in the H chain locus of C57BL6 Ig M (IgM) and do not produce mature B cells or Ab (14). SCID mice contain a spontaneous mutation in a gene encoding the catalytic subunit of DNA-activated protein kinase, resulting in the absence of B and T cells (15). TLR4-deficient mice contain a deletion of the tlr4 gene resulting in defective response to LPS stimulation (16).

Polymeric (p)IgR-deficient mice (C57BL6-pIgRtm1) were purchased from Taconic and contain a targeted deletion of the pIgR locus resulting in animals lacking secretory IgA (17). NOD1-deficient mice were obtained from Millennium Pharmaceuticals and contain a targeted mutation of CARD4. The genotype was confirmed as described previously (18). TLR2−/− and MyD88−/− mice were generated in the Akira laboratory and are previously described (19, 20).

Studies were conducted in compliance with the guidelines of the University of Pennsylvania, and all mice were housed in accordance with Institutional Animal Care and Use Committee protocols. Water and a standard rodent diet were provided ad libitum. Mice included both males and females inoculated at the age of 5 to 8 wk unless otherwise specified.

Hi strains were grown in Brain heart infusion broth (BD Biosciences) supplemented with 2% Fildes Enrichment (Remel) and 20 μg/ml ß-NAD (Sigma-Aldrich). Strains were previously described and included: H636, Eagan type b capsule (Hib); H648, a spontaneous b mutant of Eagan lacking both copies of the cap locus (21); H631, NTHI strain TN106.P2 (22); H632, NTHI strain SR7332 (23); and H680, NTHi strain 86.028NP (24). Strains H631 and H632 were used because they had been previously shown to persist in the murine airway. H680 was chosen because of the availability of its entire genomic sequence. All strains used in experiments were spontaneously streptomycin-resistant mutants and animal passaged.

Mice were inoculated intranasally with 10 μl containing 107-108 CFU of PBS-washed, mid-log phase Hi. The animal was sacrificed at the appropriate time point, the trachea cannulated, and 200 μl of PBS instilled (500 μl of PBS was used for cytospin preparation). Lavage fluid was collected from the nares for determination of viable counts of bacteria in serial dilutions plated on selective medium containing streptomycin (100 μg/ml) to inhibit the growth of contaminants. The lower limit of detection for bacteria in lavage fluid was 20 CFU/ml.

mAb RB6-8C5, a rat anti-mouse IgG2b directed against Ly6G on the surface of murine myeloid (and limited subpopulations of lymphoid) lineage cells, was purified from ascites of nude mice given the RB6-8C5 hybridoma (25, 26). To deplete neutrophils, 150 μg of mAb/animal was administered by i.p. injection 24 h before intranasal challenge with bacteria. This dose was shown in pilot experiments to result in peripheral blood neutropenia (<50 granulocytes/μl) for a period of at least 4 days. Controls were given the equivalent i.p. dose of total rat IgG (Sigma-Aldrich). Hypocomplementemia was induced by i.p. injection of 25 μg/animal of cobra venom factor (Quidel) in PBS 18 h before bacterial challenge. This procedure was previously shown to reduce levels of immunodetectible C3 to less than 3% of normal and result in a period of hypocomplementemia of greater than 48 h (27).

At the time indicated postinoculation, the animal was sacrificed and decapitated, and the head was fixed for 2 days in formalin (4% paraformaldehyde) and decalcified in 0.12 M EDTA (pH 7.0) over 30 days. The heads were then frozen in Tissue-Tek OCT embedding medium (Electron Microscopy Sciences) and 5-μm-thick sections were cut and either stained with H&E or stored at −80°C. For immunofluorescence, sections were postfixed in 1:1 methanol/acetone at −20°C for 10 min followed by washing with PBS. Nonspecific binding was inhibited by incubating for 10 min with protein-blocking reagent (Coulter-Immunotech). Sections were then incubated for 1 h at room temperature with primary Abs including polyclonal rabbit H. influenzae antiserum type b (BD Biosciences) to detect bacteria (1/400) and rat anti-mouse Ly6G to detect neutrophils (1/200). After PBS washing, secondary Abs including Cy3-conjugated donkey anti-rabbit IgG (1/400) to detect bacteria and Cy2-conjugated donkey anti-rat IgG (1:400) to detect neutrophils (Jackson ImmunoResearch Laboratories) for 1 h at room temperature. After washing with PBS followed by dH2O, sections were counterstained with 4′,6-diamidino-2-phenylindole (Molecular Probes, Invitrogen) diluted 1/10,000 in dH2O. All imaging was performed using a Nikon Eclipse E600 microscope equipped with a high-resolution charge-coupled device digital camera (CoolSNAP). All image analysis was conducted using IPLAB (Scanalytics).

Peritoneal exudate cells (PECs) were isolated from 10-wk-old C57BL6 mice as previously described (28). In brief, phagocytes were obtained by lavage of the peritoneal cavity (8 ml/animal with PBS containing 0.02 M EDTA) of mice treated 1 day and again 2 h before cell harvest by i.p. administration of 10% casein in PBS (1 ml per dose). In some experiments, where indicated, cells collected from the peritoneal cavity were enriched for neutrophils using separation in a Ficoll density gradient centrifugation using Mono-Poly Resolving Medium according to the manufacturer’s instructions (MP Biomedicals). This neutrophil-enriched fraction was collected and washed with 5 ml of Hanks’ buffer without Ca2+ or Mg2+ (Invitrogen) plus 0.1% gelatin. This population of cells was characterized previously using flow cytometric analysis for staining of granulocytes with anti-mouse Ly-6G mAb and CD11b (BD Biosciences) (18). Total PECs were collected from the peritoneal cavity of casein-treated C57BL6, TLR2−/−, and NOD1−/− mice and washed with 5 ml of Hank’s buffer without Ca2+ or Mg2+ (Invitrogen) plus 0.1% gelatin. Flow cytometric analysis was performed on PECs to characterize the relative proportions of activated neutrophils (Ly-6G+, CD11b+) and macrophages (F4/80+, CD11b+). Some mice, where indicated, were pretreated with RB6-85C mAb (as described above) before initial casein injection and isolation of total PECs.

Neutrophil-enriched or total PECs were counted by trypan blue staining and adjusted to a density of 7 × 106 cells/ml. Killing during a 45-min incubation at 37°C with rotation was assessed by combining 10 μl of 102 PBS-washed, mid-log phase bacteria preopsonized with 40 μl of a complement source, and 40 μl containing 105 murine neutrophil-enriched or total PECs, and 130 μl Hanks’ buffer with Ca2+ and Mg2+ (Invitrogen) plus 1% gelatin. The complement source consisted of fresh serum from either uninfected C57BL/6 (WT) or μMT (Ab-deficient) mice. In some cultures, complement was inactivated by heating to 56°C for 30 min before mixing with bacteria. After this reaction, viable counts were determined by plating serial dilutions. Percent killing was determined relative to identical experimental conditions without the addition of murine neutrophil-enriched or total PECs.

The nasal lavage fluid of four to five mice from each group were pooled, centrifuged at 450 × g, and resuspended in PBS containing 1% BSA. Nonspecific binding was blocked using a rat mAb against FCγIII/II receptor (CD16/CD32) and the following Abs were applied: rat anti-mouse Ly6G to detect neutrophils; rat anti-mouse CD11b, a cell surface marker of neutrophil activation; rat anti-mouse F4/80 to detect macrophages; and rat anti-mouse CD45 to detect total leukocytes. All Abs were obtained from BD Biosciences except for F4/80, which was purchased from eBioscience. A total of 10,000 cells were collected for each sample, and groups were compared using FlowJo software (Tree Star).

Lavage fluid (500 μl) was collected from 5 C57BL6 mice inoculated with Hi (day 3 post inoculation (p.i.)) and spun onto a ColorFrost Plus microscope glass slide (Fisher Scientific) using a Shandon Cytospin 3 cytocentrifuge (Thermo Shandon) at 450 × g for 10 min. The slides were briefly air-dried and subjected to differential staining using KWIK-DIFF kit (Thermo Shandon). Quantification of monocytes/macrophages was performed using standard morphological criteria. Slides were analyzed by bright-field imaging using a Nikon Eclipse E400 Microscope (Nikon Instruments) at ×400 magnification.

Hi strains were grown to mid-log phase, and chromosomal DNA was isolated by Wizard Genomic DNA Purification Kit according to the manufacturer’s protocol for Gram-negative bacteria (Promega). For detection of the genomic island that includes the T4SS expressed by some Hi strains, primer sets were designed to target two T4SS genes exhibiting homology to traB and pilT, and to target parA, a putative replication region, as previously described (29). A primer set to betT (positive control) was also designed (Forward: 5′-GCGTCGACCTGTTTCGCTATTAACCCAATT-3′; Reverse: 5′-CCCAAGATTGAAGATACAATAGTTTCAGTAAAA-3′). Portions of parA, traB, pilT, and betT were amplified with Taq Polymerase (Invitrogen) according to manufacturer’s instructions. After an initial denaturation step of 2 min at 94°C, DNA was amplified for 30 cycles, with each cycle consisting of 45 s at 94°C, 1 min at 48°C, and 1.5 min at 72°C, followed by a final extension step of 7 min at 72°C.

Statistical comparisons of colonization among groups were made by the Mann-Whitney U test (GraphPad Software) unless otherwise specified.

To investigate host and bacterial factors involved in mucosal clearance of Hi, we used a murine model of nasopharyngeal colonization. We identified an encapsulated Hib exhibiting detectable levels of colonization of C57BL6 mice by isolating bacteria from the nasal lavage fluid of infected mice. Colonization levels were examined at 1, 3, and 14 days p.i. revealing variable colonization levels at day 1, a low level of Hib colonization at day 3, and no detectable bacteria by 14 days (Fig. 1 and data not shown). Since limited colonization of Hib was observed at day 3 p.i., indicating effective clearance, this time point was used in additional experiments to evaluate the role of host factors in this process.

FIGURE 1.

Innate immunity limits Hib colonization. The density of Hi-expressing type b capsular polysaccharide in the upper respiratory tract lavage fluid of various murine strains was determined at 3 days post-intranasal inoculation. Mouse strains included WT (C57BL6-circles), SCID (no adaptive immune responses-triangles), pIgR (no secretory Ab production-squares), RB6-85C (i.p. Ab treatment to deplete neutrophil-like cells-diamonds), cobra venom factor (i.p. cobra venom factor pretreatment to deplete complement-inverse triangles), and MyD88 (no signaling via this adaptor protein-asterisks). The log CFU/ml of H636 in the lavage fluid is indicated for each mouse and horizontal bars denote geometric mean values. The dotted line indicates the limit of detection. Statistical differences were determined using the Mann-Whitney U test; ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗; p < 0.001.

FIGURE 1.

Innate immunity limits Hib colonization. The density of Hi-expressing type b capsular polysaccharide in the upper respiratory tract lavage fluid of various murine strains was determined at 3 days post-intranasal inoculation. Mouse strains included WT (C57BL6-circles), SCID (no adaptive immune responses-triangles), pIgR (no secretory Ab production-squares), RB6-85C (i.p. Ab treatment to deplete neutrophil-like cells-diamonds), cobra venom factor (i.p. cobra venom factor pretreatment to deplete complement-inverse triangles), and MyD88 (no signaling via this adaptor protein-asterisks). The log CFU/ml of H636 in the lavage fluid is indicated for each mouse and horizontal bars denote geometric mean values. The dotted line indicates the limit of detection. Statistical differences were determined using the Mann-Whitney U test; ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗; p < 0.001.

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Mucosal clearance of Hib at day 3 p.i., before the predicted onset of adaptive immune responses, suggests that innate immunity is essential. Therefore, we set forth to directly rule out a role of adaptive immune responses in clearance by inoculating SCID mice with Hib. The majority of animals were able to clear colonization by day 3 p.i. (Fig. 1). As a supplementary method of investigating the role of Ab in Hib clearance, pIgR-deficient mice were tested. These mice, who do not transport Ab to the mucosal surface, did not exhibit attenuated clearance of Hib when compared with the WT mice (Fig. 1). Cumulatively, these data suggest that innate rather than adaptive immunity is essential for mucosal clearance of Hi. Therefore, we examined the involvement of specific innate immune components, including inflammatory cells, complement, and PRMs in the clearance of colonization.

To assess whether mucosal clearance of Hib is neutrophil dependent, mice were treated with RB6-8C5, a rat mAb recognizing murine Ly6G, before intranasal challenge. This treatment has been shown to deplete neutrophils from peripheral blood (26) and prevent their recruitment to the nasopharynx in colonized mice (30). Mice treated with RB6-85C had an increased density of Hib colonization at day 3 p.i. as compared with WT (p = 0.0005), indicating attenuated clearance (Fig. 1). To determine the role of complement in clearance, mice were treated with cobra venom factor before bacterial challenge to induce hypocomplementemia. Although clearance was not attenuated in many treated mice, some mice exhibited high levels of Hib colonization (Fig. 1), suggesting that clearance may be enhanced by complement deposition.

To investigate the role of a broad range of innate immune signaling pathways in mucosal clearance, colonization of MyD88-deficient mice by Hib was analyzed. MyD88 is involved in transmitting activation signals from most TLRs and IL-1R (31). MyD88-deficient mice exhibited an increased level of Hib colonization as compared with WT mice (Fig. 1) (p < 0.0001), demonstrating attenuated bacterial clearance. Cumulatively, these data reveal a role for innate immunity, specifically neutrophils and MyD88 signaling, in mucosal clearance of Hib.

Since innate immune components are essential to the effective clearance of Hib, we characterized the inflammatory response induced by colonization by using histological examination of colonized nasal tissues. This approach demonstrated that Hib induced an influx of inflammatory cells into lateral nasal spaces by 3 h p.i. with a maximal response by 24 h p.i. (Fig. 2,A). Immunofluorescent staining of frozen tissue from 24 h-infected mice demonstrated that the infiltrate contained dense clusters of Ly6G-staining neutrophils associated with the bacteria (Fig. 2 B). To determine whether macrophages were also recruited to the site of infection, cytospins of nasal lavage fluid were prepared and monocyte/macrophage cells were enumerated. However, an average of <10 monocyte/macrophage cells were identified within the lavage fluid of each animal (data not shown).

FIGURE 2.

Hib colonization induces an acute inflammatory response including recruitment of neutrophils. A, Bacterial colonization induces an inflammatory infiltrate in the lumen of murine nasal turbinates. H&E staining of sections prepared 3 h (left panel), 24 h (center panel), or 3 days (right panel) p.i. with Hib (Hi-expressing type b capsular polysaccharide) at ×40 (top panels) and ×400 (bottom panels). Images are representative of three independent experiments with three to five mice analyzed per experiment. B, The presence of neutrophils and Hib within the infiltrate induced by bacterial colonization. At 24 h postinoculation, IF labeling (×100) of the nasal lumen and turbinates with anti-Ly6G (green) and anti-Hib capsular antiserum (red) demonstrates the association of neutrophils and bacteria. Nuclei are labeled by 4′,6-diamidino-2-phenylindole (blue). This image is representative of three independent experiments. C, Activated neutrophils kill Hib in complement-dependent, Ab-independent manner. Hib was preopsonized with sera from uninfected C57BL6 mice (WT) or Ab-deficient mice (μMT) (black bars) and where indicated, serum was incubated for 30 min at 56°C to inactivate complement (HI-hatched bars). Preopsonized bacteria were incubated with neutrophil-enriched PECs and survival of Hib was assessed over a 45-min incubation compared with non-neutrophil controls. Values are based on three or four independent determinations in duplicate + SD. Statistical differences were determined using the Paired t test; ∗, p < 0.05; ∗∗, p < 0.01.

FIGURE 2.

Hib colonization induces an acute inflammatory response including recruitment of neutrophils. A, Bacterial colonization induces an inflammatory infiltrate in the lumen of murine nasal turbinates. H&E staining of sections prepared 3 h (left panel), 24 h (center panel), or 3 days (right panel) p.i. with Hib (Hi-expressing type b capsular polysaccharide) at ×40 (top panels) and ×400 (bottom panels). Images are representative of three independent experiments with three to five mice analyzed per experiment. B, The presence of neutrophils and Hib within the infiltrate induced by bacterial colonization. At 24 h postinoculation, IF labeling (×100) of the nasal lumen and turbinates with anti-Ly6G (green) and anti-Hib capsular antiserum (red) demonstrates the association of neutrophils and bacteria. Nuclei are labeled by 4′,6-diamidino-2-phenylindole (blue). This image is representative of three independent experiments. C, Activated neutrophils kill Hib in complement-dependent, Ab-independent manner. Hib was preopsonized with sera from uninfected C57BL6 mice (WT) or Ab-deficient mice (μMT) (black bars) and where indicated, serum was incubated for 30 min at 56°C to inactivate complement (HI-hatched bars). Preopsonized bacteria were incubated with neutrophil-enriched PECs and survival of Hib was assessed over a 45-min incubation compared with non-neutrophil controls. Values are based on three or four independent determinations in duplicate + SD. Statistical differences were determined using the Paired t test; ∗, p < 0.05; ∗∗, p < 0.01.

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To determine whether murine neutrophils, which were recruited to the nasal spaces in response to Hib colonization, are involved in its elimination, neutrophils enriched from elicited PECs were analyzed in ex vivo killing assays. Neutrophil-enriched PECs were incubated with preopsonized Hib. Hib-killing was observed when bacteria were preopsonized with sera isolated from uninfected C57BL6 mice (Fig. 2,C). Killing was independent of the presence of specific Ab since serum isolated from Ab-deficient mice (μMT) resulted in equivalent levels of Hib killing. Heat-inactivation of sera before opsonization resulted in minimal neutrophil-enriched PEC-mediated Hib killing, indicating complement-dependence. Cumulatively, these data reveal the recruitment of neutrophils to the site of Hib colonization (Fig. 2, A and B) that is essential for mucosal clearance (Fig. 1). Neutrophils were associated with the bacteria (Fig. 2,B) and capable of killing Hib ex vivo (Fig. 2,C). Moreover, the neutrophilic infiltrate is no longer present 3 days p.i. (Fig. 2,A), corresponding with the low level of residual bacterial colonization appreciated at this time point (Fig. 1).

Since MyD88-deficient mice exhibited attenuated bacterial clearance (Fig. 1), we set forth to further define which individual MyD88-dependent signaling pathways may be involved in the mucosal clearance of Hib. This was accomplished by examining the role of the specific pattern recognition receptors, TLR4 and TLR2, by use of TLR4- and TLR2-deficient mice. Additionally, the role of the pattern recognition molecule NOD1 was also investigated using NOD1-deficient mice. Mice deficient in TLR2, TLR4, or NOD1 expression showed increased levels of bacterial colonization as compared with WT mice at day 3 p.i. indicating a role for each receptor pathway in mucosal clearance of Hib (Fig. 3,A) (p < 0.0001; p = 0.002; p = 0.0001, respectively). Moreover, unlike WT mice, colonization of these immune-deficient mice persisted for at least 14 days p.i. (Fig. 3,B). Contrary to what was observed for TLR2 and NOD1-deficient mice, the density of colonizing Hib in TLR4−/− mice significantly increased between days 3 and 14 p.i. (p = 0.002; Fig. 3 B), suggesting that the mucosal clearance of these mice remained attenuated even after the expected time frame for the initiation of adaptive immune defenses. Cumulatively, these data demonstrate that the absence of expression of each individual signaling pathway was sufficient to result in increased and more persistent Hib colonization.

FIGURE 3.

Expression of TLR2, NOD1, and non-neutrophil TLR4 are essential for clearance of Hib. A, TLR2−/− (triangles), TLR4−/− (squares), and NOD1−/− (diamonds) mice exhibited attenuated clearance compared with WT controls at day 3 p.i. with Hi-expressing type b capsular polysaccharide. Where indicated, mice were pretreated 24 h before inoculation with mAb RB6-85C to deplete neutrophils (open symbols). The horizontal bars indicate geometric mean values and the log CFU/ml of bacteria is indicated for each mouse. B, Colonization of C57BL6 (WT), TLR2−/−, TLR4−/−, and NOD1−/− mice by Hib at 3 (black bars; WT = 32 mice, TLR2−/− = 5 mice, TLR4−/− = 7 mice, NOD1−/− = 9 mice) and 14 (diagonal bars; WT = 5 mice, TLR2−/− = 5 mice, TLR4−/− = 5 mice, NOD1−/− = 7 mice) days post inoculation. Density of Hi in upper respiratory tract lavage fluid is shown by the mean log CFU/ml + SD. The dotted line indicates the limit of detection. Statistical differences were determined using the Mann-Whitney U test: ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001. C, WT mice recruited activated neutrophils to the site of infection following Hib inoculation. Flow cytometric analysis demonstrating the percentage of cells recovered from the nasal lavage fluid that express both Ly6G (neutrophil) and CD11b (activation) markers (top right of panel) from WT mice. Image is representative of five independent experiments. D, TLR2−/−, NOD1−/−, and TLR4−/− mice recruited neutrophils to the site of infection following Hib inoculation. Percent of Ly6G+ cells per 10,000 events evaluated from nasal lavage fluid of WT (20 mice), TLR2−/− (10 mice), NOD1−/− (20 mice), and TLR4−/− (10 mice) mice 24 h post Hib-inoculation as determined by flow cytometric analysis. Values are based on the mean + SD. E, Phagocytes from NOD1-deficient mice did not effectively kill Hib. Total PEC phagocytes were obtained from WT, TLR2−/−, and NOD1−/− mice following i.p. administration of casein. Phagocytes were incubated with H636 preopsonized with sera from uninfected C57BL6 mice for 45 min, and survival was assessed compared with non-phagocyte control groups. No stimulation of killing was observed in controls using heat-inactivated sera. Values are based on three or four independent determinations in duplicate + SD. Statistical differences were determined using the Paired t test; ∗, p < 0.05; ∗∗, p < 0.01.

FIGURE 3.

Expression of TLR2, NOD1, and non-neutrophil TLR4 are essential for clearance of Hib. A, TLR2−/− (triangles), TLR4−/− (squares), and NOD1−/− (diamonds) mice exhibited attenuated clearance compared with WT controls at day 3 p.i. with Hi-expressing type b capsular polysaccharide. Where indicated, mice were pretreated 24 h before inoculation with mAb RB6-85C to deplete neutrophils (open symbols). The horizontal bars indicate geometric mean values and the log CFU/ml of bacteria is indicated for each mouse. B, Colonization of C57BL6 (WT), TLR2−/−, TLR4−/−, and NOD1−/− mice by Hib at 3 (black bars; WT = 32 mice, TLR2−/− = 5 mice, TLR4−/− = 7 mice, NOD1−/− = 9 mice) and 14 (diagonal bars; WT = 5 mice, TLR2−/− = 5 mice, TLR4−/− = 5 mice, NOD1−/− = 7 mice) days post inoculation. Density of Hi in upper respiratory tract lavage fluid is shown by the mean log CFU/ml + SD. The dotted line indicates the limit of detection. Statistical differences were determined using the Mann-Whitney U test: ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001. C, WT mice recruited activated neutrophils to the site of infection following Hib inoculation. Flow cytometric analysis demonstrating the percentage of cells recovered from the nasal lavage fluid that express both Ly6G (neutrophil) and CD11b (activation) markers (top right of panel) from WT mice. Image is representative of five independent experiments. D, TLR2−/−, NOD1−/−, and TLR4−/− mice recruited neutrophils to the site of infection following Hib inoculation. Percent of Ly6G+ cells per 10,000 events evaluated from nasal lavage fluid of WT (20 mice), TLR2−/− (10 mice), NOD1−/− (20 mice), and TLR4−/− (10 mice) mice 24 h post Hib-inoculation as determined by flow cytometric analysis. Values are based on the mean + SD. E, Phagocytes from NOD1-deficient mice did not effectively kill Hib. Total PEC phagocytes were obtained from WT, TLR2−/−, and NOD1−/− mice following i.p. administration of casein. Phagocytes were incubated with H636 preopsonized with sera from uninfected C57BL6 mice for 45 min, and survival was assessed compared with non-phagocyte control groups. No stimulation of killing was observed in controls using heat-inactivated sera. Values are based on three or four independent determinations in duplicate + SD. Statistical differences were determined using the Paired t test; ∗, p < 0.05; ∗∗, p < 0.01.

Close modal

Interestingly, when TLR4−/− mice were depleted of neutrophils before bacterial inoculation, the observed colonization levels of Hib were increased as compared with control TLR4−/− mice (p = 0.0002; Fig. 3,A). These data reveal an additive effect of non-neutrophil TLR4 expression and neutrophil influx on the mucosal clearance of Hib. Conversely, depletion of neutrophils from TLR2 and NOD1-deficient mice before Hib inoculation did not result in an additional increase in colonization levels as compared with respective knockout mice without neutrophil depletion (Fig. 3 A). Therefore, Hib stimulation of TLR2 and/or NOD1 may act on the same pathway as neutrophils to induce mucosal clearance. In contrast, the greater effect of TLR2 or NOD1 deficiency compared with neutrophil depletion may indicate that these pattern recognition proteins also function on non-neutrophil cell populations.

To determine whether neutrophil-recruitment to the site of infection is observed in Hib-colonized TLR2-deficient and NOD1−/− mice, flow cytometric analysis was performed on cells isolated from the nasal lavage fluid. A population of Ly6G+ and CD11b+ expressing cells were isolated from WT mice, confirming the presence of activated neutrophils within the nasal spaces of colonized mice (Fig. 3,C) and supporting the immunofluorescence data presented previously (Fig. 2,B). Similar to what was observed for the Hib-colonized WT mice, the majority of cells isolated from nasal lavage of colonized TLR2−/−, TLR4−/− and NOD1−/− mice coexpressed the markers Ly6G and CD11b (Fig. 3,D and data not shown). The neutrophil response was more marked in immunodeficient mice, suggesting there may be dysregulation of recruitment (Fig. 3 D). Cumulatively, this confirmed that deficiencies in TLR2, TLR4, or NOD1 did not limit the accumulation of activated neutrophils at the site of colonization.

Since TLR2 and NOD1 expression were not essential to the recruitment and activation of neutrophils at the site of infection, we examined the role of these recognition proteins in the phagocytic killing of Hib. To investigate phagocytic killing without limiting analysis to a specific cell type, elicited PECs (without neutrophil enrichment) were isolated from the murine peritoneal cavity of WT, TLR2−/−, and NOD1−/− mice. Flow cytometric analysis of these exudates indicated that the majority of CD45+ cells were activated neutrophils (Ly6G+ and CD11b+) and macrophages (F4/80+ and CD11b+) (data not shown). Incubation of WT phagocytes with preopsonized Hib resulted in complement-dependent bacterial killing. Pretreatment of mice with RB6-8C5 before isolation of total PECs to deplete Ly6G+ cells resulted in no PEC-mediated bacterial killing (data not shown). This provides further evidence that Hib killing by total PECs is mediated by activated neutrophils; either by direct phagocytic activity and/or signaling involved in the recruitment of other phagocytic cells. Phagocytes isolated from TLR2-deficient mice exhibited less killing of Hib as compared with WT (p = 0.04; Fig. 3,E), demonstrating that expression of this pathway aids in effective phagocytic killing of encapsulated Hi. Moreover, no bacterial killing was observed with phagocytes isolated from NOD1-deficient mice (Fig. 3 E), indicating that expression of this pathway is required for killing of the encapsulated Hi strain by phagocytic cells.

To investigate the role of polysaccharide capsule expression in mucosal clearance of Hi, we first attempted to identify nonencapsulated strains (NTHi) that exhibit detectable levels of colonization of C57BL6 mice (Fig. 4 A). At day 3 p.i., low levels of one nonencapsulated strain (H631) were isolated from the nasal lavage fluid of infected mice. These levels were similar to that observed for Hib (H636) and indicate stable colonization of these strains. No detectable colonization of the noncapsulated strains, H632 and H680, was observed. Therefore the nonencapsulated strain, H631, was used in additional experiments to determine whether expression of polysaccharide capsule alters the requirement of various innate immune signaling pathways to promote effective mucosal clearance.

FIGURE 4.

TLR4, TLR2, and NOD1 are not required for clearance of nonencapsulated Hi. A, The density of various Hi strains in the upper respiratory tract lavage fluid was determined at 3 days post-intranasal inoculation. The Hi strains used to inoculate C57BL6 mice include H636 (Hi-expressing type b capsular polysaccharide-circles), H631 (nontypeable Hi isolate-triangles), H632 (nontypeable Hi isolate-diamonds), and H680 (nontypeable Hi isolate-asterisks). The horizontal bars indicate geometric mean values, and the log CFU/ml of bacteria is indicated for each mouse. B, The density of various Hi strains in the upper respiratory tract lavage fluid of WT (triangles), TLR2−/− (circles), TLR4−/− (diamonds), NOD1 (asterisks), and RB6-85C treated (X’s) mice was determined at 3 days post-intranasal inoculation. The Hi strains used to inoculate mice include H631 (nontypeable Hi) and isogenic strains H636 (b+) and H648 (b−). The horizontal bars indicate geometric mean values and the log CFU/ml of bacteria is indicated for each mouse.

FIGURE 4.

TLR4, TLR2, and NOD1 are not required for clearance of nonencapsulated Hi. A, The density of various Hi strains in the upper respiratory tract lavage fluid was determined at 3 days post-intranasal inoculation. The Hi strains used to inoculate C57BL6 mice include H636 (Hi-expressing type b capsular polysaccharide-circles), H631 (nontypeable Hi isolate-triangles), H632 (nontypeable Hi isolate-diamonds), and H680 (nontypeable Hi isolate-asterisks). The horizontal bars indicate geometric mean values, and the log CFU/ml of bacteria is indicated for each mouse. B, The density of various Hi strains in the upper respiratory tract lavage fluid of WT (triangles), TLR2−/− (circles), TLR4−/− (diamonds), NOD1 (asterisks), and RB6-85C treated (X’s) mice was determined at 3 days post-intranasal inoculation. The Hi strains used to inoculate mice include H631 (nontypeable Hi) and isogenic strains H636 (b+) and H648 (b−). The horizontal bars indicate geometric mean values and the log CFU/ml of bacteria is indicated for each mouse.

Close modal

The observation that individual expression of TLR2, TLR4, and the NOD1 signaling pathways was required for effective mucosal clearance of Hib was unexpected. To determine whether this phenomenon was specific to this encapsulated strain, the role of TLR2, TLR4, and NOD1 individually in the clearance of an isogenic unencapsulated mutant strain (b) and an unrelated nonencapsulated strain (NTHi) was investigated (Fig. 4 B). Like the Hib strain (b+), both the unencapsulated mutant strain and a nonencapsulated strain showed limited colonization of WT mice. Contrary to what was observed with the encapsulated Hib strain, individual deficiencies in these recognition proteins were not sufficient to attenuate clearance of nonencapsulated strain H631. Similarly, no increase in colonization of the unencapsulated mutant (b) of H636 was detected for mice deficient in TLR2, TLR4, or NOD1. Moreover, unlike the Hib strain, neutrophil depletion did not result in an increase in the colonization of the unencapsulated mutant strain or nonencapsulated strain. These data suggest the cumulative expression of TLR4, TLR2, and NOD1 recognition pathways and the requirement of neutrophils are essential for mucosal clearance of capsule-expressing, but not capsule-deficient, Hi.

In this study, we investigated host immune components required for clearance of bacteria expressing capsular polysaccharide in vivo. We determined that the mucosal clearance of capsule-expressing Hib required the function of both TLR and NOD pathways, whereas individual deficiencies in each of these signaling cascades did not affect clearance of nonencapsulated Hi. Moreover, recruitment of neutrophils to the site of infection was essential for clearance of encapsulated Hib and ex vivo phagocytic killing of Hib required expression of the NOD1 signaling pathway. Cumulatively, these data suggest a model in which both encapsulated and unencapsulated strains likely elicit innate immune pathways by similar mechanisms; however, capsule-expression provides an advantage to this organism by altering the requirements for host signaling pathways that effectively limit prolonged colonization. It is unlikely that capsular polysaccharide directly affects recognition by PRMs, since capsule expression impacts the effectiveness of three distinct signaling pathways involving TLR2, TLR4, and NOD1. A further implication is that redundancies within these innate immune pathways are sufficient to promote effective mucosal clearance of bacteria that do not express capsular polysaccharide and do not require phagocytic killing.

Cross-talk and redundant responses among TLR2 and TLR4 recognition receptors has been previously described (32, 33, 34) and encapsulated Hib has been shown to mediate activation of both pathways in vitro (35). TLR4-mediated pulmonary clearance of Hib and neutrophil influx to the lung has also been described (36). We found that clearance of encapsulated Hib was dependent upon expression of both TLR2 and TLR4 signaling pathways, each with distinct contributions to innate immunity. The absence of TLR4 expression resulted in an attenuation of clearance that was amplified by the depletion of neutrophils. In a separate study, we show that TLR4 appears to be particularly important in early epithelial responses to colonizing Hi (C. Beisswenger, E. S. Lysenko, and J. N. Wesier, manuscript submitted). In contrast, for TLR2, data in this report indicate that a major contribution to clearance may be in the activity but not recruitment of luminal neutrophils. PECs derived from TLR2−/− mice have a reduced ability to kill Hib ex vivo, indicating that neutrophil-mediated killing is enhanced by expression of this signaling pathway.

Findings in our study also emphasize the differing requirements for signaling to protect against pathogens that do not require clearance by professional phagocytes. Although a complex interaction between TLR2 and TLR4 that regulates expression and function of these receptors in response to nonencapsulated Hi infection has been suggested (37), in the mouse model of colonization their roles appear to be redundant. Early recognition of nonencapsulated Hi by activation of either of these signaling pathways in non-neutrophil cells may be sufficient to promote effective clearance. Since NTHi commonly colonizes humans, either these pathways are less effective or the organism has evolved mechanisms to evade their effects in its natural host.

In addition to cross-talk between different TLR receptors, cumulative responses among NOD and TLR innate immune signaling pathways leading to NFκB activation and response amplification has also been characterized (38, 39, 40). Therefore, it is unexpected that inactivation of single pattern recognition pathways resulted in the selective ability of Hi-expressing capsular polysaccharide to thwart host defense mechanisms and promote stable colonization within the murine nasopharynx. Moreover, it is interesting that this typical extracellular pathogen (Hib) can evoke the intracellular NOD1 signaling pathway, resulting in mucosal clearance.

Although previous studies have identified a role for NOD1-mediated effects of bacteria and cell wall components in vitro (for review see Refs. 41, 42), our understanding of the contribution of this PRM with regard to innate immune responses to infection in vivo is still incomplete. In our system, NOD1 was not a requirement for recruitment of neutrophils to the site of infection. Instead, NOD1 signaling enhanced the phagocytic activity of cell populations similar to those found recruited to murine nasal spaces following Hib inoculation. These data are supported by a previous study demonstrating that the NOD1 signaling pathway can respond to synthetic meso-diaminopimelic acid containing compounds or peptidoglycan of Hi to enhance the opsonophagocytic killing of another encapsulated bacterial pathogen, S. pneumoniae (18), further suggesting a role for NOD1 signaling in clearance of encapsulated bacteria. Characterization of the NOD1-dependent neutrophil antimicrobial activity that impacts killing of encapsulated bacteria is the subject of on-going investigation.

The mechanism of delivery of the bacterial peptidoglycan components to the intracellular compartment where NOD1 is located in this system remains unknown, although access to these cytoplasmic pathways may not be limited for professional phagocytes. It has been shown that the type IV secretion apparatus expressed by the Helicobacter pylori cag pathogenicity island mediates delivery of peptidoglycan to the host cell cytoplasm (43). Since expression of a putative type IV secretion system (T4SS) within the genomic island ICEHin1056 has recently been described for Hi (44), we determined whether the Hib strain used in our experiments expressed this T4SS. However, PCR amplification of T4SS-specific and island-specific genes was not observed for H636, suggesting that T4SS-mediated delivery of peptidoglycan is unlikely to account for NOD1 activation in our experiments (data not shown). Alternatively, production of a bacterial pore-forming toxin has been shown to permit intracellular access of peptidoglycan fragments (45). The expression of such toxins has not been described for Hi; although the possibility that production of these proteins by other members of microbial flora permits ligand access to the host cell cytoplasm cannot be excluded. In this regard, one potential explanation for the increase in Hib colonization of TLR2, TLR4, and NOD1-deficient mice is that these immune-deficient strains carry an altered microbial flora that results in an environment more permissive for Hi colonization. However, the fact that an increase in colonization was not observed universally for all Hi strains and that mice lacking effective adaptive immune responses exhibited effective clearance of Hib, suggest that these results are specific to innate immune recognition of encapsulated bacteria.

This study also indicates a direct role for neutrophil recruitment and/or activity in preventing extended colonization of encapsulated Hib. In our system, we examined the role of neutrophils in bacterial clearance by pretreating mice with the mAb RB6-85C to deplete cells expressing Ly-6G. A potential limitation of this method is that this Ab also recognizes non-neutrophil subpopulations of monocytes, dendritic cells, and macrophages (46). Cytospins, IF, and flow cytometric analysis, however, confirmed that the vast majority of cells recruited to the nasal spaces of Hib-infected mice were neutrophils. Moreover, flow cytometry demonstrated the absence of monocytes (CD14+) or dendritic cells (CD11c+) in the nasal lavage fluid of Hib-infected mice, making it unlikely that results based on depletion with this mAb could be explained by effects on these other cell types.

As is the case for many common human pathogens, readily accessible animal models of Hi infection have been limiting. We demonstrate that mice deficient in components of innate immunity can be used to study its host-pathogen interactions. In summary, these observations provide evidence that the cumulative expression of multiple innate immune signaling pathways is needed to control mucosal infection by bacteria expressing capsular polysaccharide.

We thank Bruce Green and Robert Munson for providing strains. We additionally acknowledge Kathryn Matthias and Zhe Zhang for technical expertise and assistance.

The authors have no financial conflict of interest.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

This work was supported by the U. S. Public Health Service Grants AI44231 and AI38446 to J.N.W. and Center Grant P30 DK50306 from the Molecular Studies of Liver and Digestive Diseases to the Morphology Core of the Center.

3

Abbreviations used in this paper: Hi, Haemophilus influenzae; Hib, Hi type b; PRM, pattern recognition molecule; PEC, peritoneal exudate cell; WT, wild type; p, polymeric; p.i., post inoculation.

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