Cellular effectors mediating Th17-dependent clearance of pneumococcal colonization in mice

Microbial colonization of mucosal surfaces may be an initial event in the progression to disease, and it is often a transient process. For the extracellular pathogen Streptococcus pneumoniae studied in a mouse model, nasopharyngeal carriage is eliminated over a period of weeks and requires cellular rather than humoral immunity. Here, we demonstrate that primary infection led to TLR2-dependent recruitment of monocyte/macrophages into the upper airway lumen, where they engulfed pneumococci. Pharmacologic depletion of luminal monocyte/macrophages by intranasal instillation of liposomal clodronate diminished pneumococcal clearance. Efficient clearance of colonization required TLR2 signaling to generate a population of pneumococcal-specific IL-17–expressing CD4⁺ T cells. Depletion of either IL-17A or CD4⁺ T cells was sufficient to block the recruitment of monocyte/macrophages that allowed for effective late pneumococcal clearance. In contrast with naive mice, previously colonized mice showed enhanced early clearance that correlated with a more robust influx of luminal neutrophils. As for primary colonization, these cellular responses required Th17 immunity. Our findings demonstrate that monocyte/macrophages and neutrophils recruited to the mucosal surface are key effectors in clearing primary and secondary bacterial colonization, respectively.

Acute respiratory infection remains a leading cause of mortality worldwide. Recent experience in the developing world has revealed the importance of immunization against a single pathogen, Streptococcus pneumoniae (the pneumococcus), in lowering the incidence of respiratory tract infection and overall childhood mortality (1). As is the case for many mucosal pathogens, colonization not only precedes all pneumococcal diseases but also serves as a prerequisite for the spread of the organism within the community (2–4). Longitudinal carriage studies have shown that colonization of the upper respiratory tract is a dynamic process, with most children colonized serially with single or even multiple serotypes of the pneumococcus (5). Each colonization event may persist from days to months (6). Host factors that lead to the clearance of the bacterium from its commensal niche on the mucosal surface of the human nasopharynx are not well defined.

Prior studies have emphasized the importance of humoral immunity in protection from this predominantly extracellular pathogen (7). Antibodies to pneumococcal capsular polysaccharides define the 91 known serotypes, are opsonic, and may confer serotype-specific protection. Systemic immunization with capsular polysaccharide–based vaccines induces high levels of serum antibody that are sufficient to prevent the acquisition of strains in a serotype-specific manner (8–10). However, in experimental human carriage studies, colonization generates minimal anticapsular antibody, and clearance is not temporally associated with the development of serotype-specific immunity (6, 11). Moreover, the overall decrease in rates of pneumococcal carriage that occurs with increasing age beyond early childhood occurs in a largely serotype-independent manner (12). In model pneumococcal colonization of mice, clearance of bacteria from the upper airway requires CD4⁺ but not CD8⁺ T cells and is independent of antibody (11, 13–15). Trzcinski et al. have recently confirmed that antigen-specific T cell immunity is sufficient to protect against pneumococcal colonization in mice (16). These observations suggest that cellular rather than humoral immunity may be necessary for natural immunity that promotes the clearance of pneumococcal colonization. In this regard, the depletion of CD4⁺ T cells in HIV-positive children could account for their elevated rates of pneumococcal carriage and disease (17).

Additional studies using genetic mouse models show that clearance of colonization is delayed in the absence of TLR2, which promotes signaling in leukocytes and other cells in response to lipid-modified pathogen-associated molecular patterns (18–20). Similarly, mice with a mutation affecting TLR4 signaling show diminished responses to this pathogen’s only known toxin, pneumolysin, and are more susceptible to pneumococcal colonization following nasopharyngeal challenge (21). Pneumolysin has also been implicated as a factor triggering the migration and activation of CD4⁺ T cells (22). The recognition of a TLR- and CD4⁺ T cell–dependent immune mechanism has left in question the nature of the effector(s) mediating antibody-independent clearance. Malley et al. have shown that depletion of IL-17 blocks the protective effect of mucosal immunization with a killed whole-cell vaccine, indicating the importance of the Th17 subset of CD4⁺ T helper cells (14, 15, 23).

Pneumococcal colonization induces an acute inflammatory response, with a predominance of neutrophils in the nasal spaces during initial colonization (<72 hours) (18). The antimicrobial activity of luminal neutrophils contributes to the processing of bacterial antigen and its delivery to the nasal-associated lymphoid tissue, where adaptive immunity may be initiated (24). However, this early influx of neutrophils does not correlate with the decline in the density of pneumococci during colonization, which requires up to several weeks following primary challenge, suggesting that neutrophils may not be the main effectors of clearance (11).

In this report, we characterize the cellular effectors controlling the clearance of mucosal colonization by an extracellular bacterial pathogen. We demonstrate a TLR2-, IL-17A-, and CD4⁺ T cell–dependent recruitment of monocyte/macrophages (primary and secondary colonization) and neutrophils (secondary colonization) into the lumen of the upper airway. Furthermore, we document the contribution of these cells to the clearance of pneumococci from the mucosal surface.

Monocyte/macrophages promote clearance of colonization. We investigated the cellular immune responses in murine colonization with strain P1121. This type 23F clinical isolate was previously characterized in experimental human carriage and colonizes the nasopharynx of C57BL/6 mice for a similar duration with a similar immune response (5, 6). Upper respiratory tract lavages were used to evaluate the kinetics of colonization by quantitative culture. The cellular inflammatory response to colonization was characterized in lavages by differential cell quantification on cytospin preparations. As previously documented, the density of P1121 decreased gradually over the observation period and was below the limit of detection (10 CFUs/animal) by day 28 after inoculation (Figure 1A and data not shown) (11). Numbers of luminal neutrophils were maximal at day 3 after inoculation and thereafter declined to precolonization levels by day 21 after inoculation (Figure 1B). Luminal mononuclear cells, present in precolonized mice in low numbers (<10 cells/animal), reached peak numbers at day 7 after inoculation and remained elevated throughout the remainder of the colonization period (Figure 1C). The mononuclear cells were confirmed to be monocyte/macrophages by staining with monocyte/macrophage marker (MOMA-2) mAbs, which did not bind neutrophils (Figure 1D). Of note, many of the MOMA-2–positive cells had vacuole structures that are typical of activated macrophages (Figure 1D). Recruitment of monocyte/macrophages was confirmed by flow cytometric analysis on nasal lavages pooled from 5 mice at day 7 after inoculation (Figure 1E). The majority of CD45⁺ events in nasal lavages stained positively with macrophage marker Ly6C (Figure 1E). Many of the cells also costained with the monocyte marker CD14 (data not shown).

Figure 1

Macrophages are recruited to nasopharynx in association with clearance of pneumococcal isolate P1121, and responses are attenuated in the absence of TLR2 signaling. Upper respiratory tract lavages were analyzed at the time indicated following i.n. inoculation of C57BL/6 WT mice (filled symbols) and congenic Tlr2^–/– mice (open symbols) for quantitative culture to determine the time course of P1121 colonization (A). Cells in cytospin preparations of colonized mice were used to determine the time course of (B) neutrophil and (C) mononuclear cell recruitment. Animals at day 0 were mock colonized. n = 5 to 20 mice per group per time point. Values represent means ± SEM. (D) MOMA-2–positive mononuclear cells are recruited to the lumen of the nasopharynx. MOMA-2 mAb staining (red) of mononuclear cells in the cytospin preparations of nasal lavages from mice 7 days after P1121 challenge with or without Nomarski optics. Isotype-matched antibody was used as a negative control (Neg.). Nuclei were stained with DAPI (blue). Lower right panel shows a mononuclear cell (arrow) and olfactory epithelium lining the nasal cavity in a tissue section stained with H&E. Original magnification, ×1000 (upper panels); ×400 (lower left panel); ×200 (lower right panel). (E) Quantification by flow cytometry showing a representative experiment comparing the number of infiltrating macrophages in pooled nasal lavages from 5 WT or Tlr2^–/– mice at day 21 of P1121 colonization. Naive mice were mock inoculated with PBS. The numbers in the upper left corners indicate the number of Ly6C⁺ and CD45⁺–double-positive cells in the sample. **P < 0.01; ***P < 0.001.

Since the steady decline in the density of colonizing P1121 correlated with the presence of luminal monocyte/macrophages, we addressed whether these cells promote clearance. Liposomes encapsulating clodronate (Cl₂MDP) have been used widely to eliminate monocytes and macrophages in the blood, spleen, and liver (24). i.n. administration of Cl₂MDP liposomes led to a more than 80% decline in the number of monocyte/macrophages compared with liposome-alone controls as assessed in cytospin preparations at both day 7 and day 28 after inoculation (P = 0.0003 and 0.01, respectively; Figure 2A). There was no significant effect of liposomes with or without Cl₂MDP on numbers of luminal neutrophils (data not shown). The liposome preparations did not show any significant toxicity to P1121 growth in broth cultures (data not shown). Efficiency of the monocyte/macrophage depletion was also evident by flow cytometric analysis of CD45⁺– and Ly6C⁺–double-positive events in nasal lavages (Figure 2C). In association with the depletion of monocyte/macrophages, there was diminished clearance of S. pneumoniae at both day 7 and day 28 after inoculation (P = 0.03 and P = 0.001, respectively; Figure 2B).

Figure 2

Monocyte/macrophage influx is required for clearance of pneumococcal strain P1121. Mice were administered liposome-Cl₂MDP or liposome alone, and the effect of depletion was evaluated at day 7 or 28 after bacterial inoculation as described in Methods. (A) Depletion of mononuclear cells in cytospin preparations as evaluated by differential cell quantification. Closed symbols, liposome; open symbols, liposome-Cl₂MDP. (B) Effect of monocyte/macrophage depletion on the density of colonization by strain P1121.Values represent individual animals, with the mean shown by a horizontal bar. (C) Quantification of the depletion of monocyte/macrophages at day 7 by flow cytometry. The numbers of CD45⁺– and Ly6C⁺–double-positive events in pooled nasal washes (n = 5 per group) at day 7 of P1121 colonization compared with that of naive/PBS mock-colonized control are shown. Mice were administered liposome-Cl₂MDP (lower panel) or liposome alone as control (upper panel). *P < 0.05; **P < 0.01; ***P < 0.001.

Next, we determined whether there is a physical association between bacteria and these cells. Confocal microscopy following immunostaining for both monocyte/macrophages and strain P1121 on nasal lavage cytospin preparations from day 7 after inoculation showed that the pneumococci were commonly engulfed by macrophage-like cells (Figure 3A). No similar association between pneumococci and neutrophils was observed (Figure 3A). In addition, bacteria associated with macrophage-like cells costained with the phagolysosome marker lysosome-associated membrane protein-1 (LAMP-1) (Figure 3B). Together, these findings indicate that bacterial colonization leads to the recruitment of monocyte/macrophages that directly associate with pneumococci and contribute to their clearance following phagocytosis.

Figure 3

Macrophages in the lumen of the upper respiratory tract engulf colonizing pneumococci. (A) MOMA-2 mAb staining–positive (red) mononuclear cells associated with pneumococci stained with type 23F antiserum (green). Left panel shows a representative z-section confocal image. Cytospin preparations were from mice inoculated with P1121 for 7 days. Scale bar: 5 μm. Right panel shows representative view of a cytospin preparation from mice inoculated with P1121 for 3 days. Pneumococci were observed to be associated with monocytes/macrophages but not neutrophils. Original magnification, ×200. (B) P1121 stained with type 23F antiserum (green) colocalized with LAMP-1–positive (red) intracellular lysosomal compartments as shown in the overlay (yellow). Mononuclear cells were identified by DAPI staining of the nucleus (blue). Cytospin preparations were from mice inoculated with P1121 for 7 days. Original magnification, ×600.

Host factors contributing to clearance by monocyte/macrophages during primary infection. The role of TLRs was investigated using KO mice. As previously reported, Tlr2^–/– mice were unaffected in early colonization (day 7) but showed delayed clearance at day 21 after inoculation (P = 0.007; Figure 1A) (18). These mice showed a more prolonged influx of neutrophils but no difference in the number of these cells by day 21 after inoculation when defective clearance is observed (Figure 1B). Rather, at this time point and despite an increased number of bacteria, Tlr2^–/– mice showed impaired recruitment of monocyte/macrophages (P = 0.0004; Figure 1C). The attenuated recruitment of CD45⁺– and Ly6C⁺–double-positive cells in colonized Tlr2^–/– mice was confirmed by flow cytometry on pooled nasal lavages (Figure 1E). In contrast, for TLR4 KO mice (C57BL/10ScNJ), colonization levels of P1121 and monocyte/macrophage recruitment were comparable to those of congenic WT mice (data not shown). These observations suggest that TLR2 but not TLR4 signaling is required for the recruitment of monocyte/macrophages that mediate clearance.

Likewise, depletion of CD4⁺ T cells had no effect on early colonization (day 7; data not shown) but resulted in delayed late clearance at day 21 after inoculation (P = 0.000002; Figure 4A). The CD4⁺ T cell–dependent inhibition of clearance was associated with a decrease in the recruitment of monocyte/macrophages (P = 0.005; Figure 4C). There was no effect of CD8⁺ T cell depletion on the density of bacteria or monocyte/macrophage recruitment. Depletion of either CD4⁺ or CD8⁺ T cells showed no effect on neutrophil responses (Figure 4B).

Figure 4

Cytokine IL-17A and CD4⁺ but not CD8⁺ T cells are required for strain P1121 clearance and monocyte/macrophage recruitment. Mice were administered isotype control antibody (closed circles), IL-17A neutralizing antibody (open circles), 2.43 antibodies to deplete CD8⁺ T cells (open triangles), or GK1.5 antibodies to deplete CD4⁺ T cells (closed triangles) as described in Methods. Nasal lavages were assessed at day 21 after bacterial challenge for (A) density of P1121 colonization, (B) neutrophil recruitment, and (C) monocyte/macrophage recruitment. n = 5–20 mice per group per time point. Values represent individual animals, with mean shown by a horizontal bar. *P < 0.05; **P < 0.01; ***P < 0.001.

Next, we determined whether the contribution of CD4⁺ T cells to monocyte/macrophage-mediated clearance required a Th17 response. Systemic depletion of IL-17 during primary colonization using an antibody to murine IL-17A (administered at days 7 and 14 after inoculation) resulted in a loss of bacterial clearance at day 21 after inoculation (P = 2 × 10^–6; Figure 4A). Attenuated clearance in anti–IL-17A–treated mice was associated with a complete attenuation of monocyte/macrophage recruitment (P = 0.01; Figure 4C).

It was concluded that the effects of TLR2, CD4⁺ T cells, and IL-17A on the recruitment of monocyte/macrophages into the airway lumen could account for their previously described role in the clearance of pneumococcal colonization (11, 14, 18).

Host factors contributing to clearance during secondary infection. Our model of clearance in naive mice predicted that in immune mice, there would be an existing population of pneumococcal-specific IL-17–expressing CD4⁺ T cells. Cellular responses in primed mice should, in turn, facilitate a more rapid recruitment of phagocytes and clearance. To test this prediction, mice were first challenged i.n. with 10⁷ CFUs of strain P1121, and 6 weeks later, after confirmation of complete nasopharyngeal clearance in pilot experiments, each mouse was given a second i.n. dose of 10⁷ CFUs of strain P1121. Analysis of splenocytes from previous colonized mice showed expansion of a population of CD4⁺ T cells expressing IL-17 in response to pneumococcal stimulation (Figure 5A). This expansion of IL-17–positive cells in splenocytes from previously colonized mice in response to pneumococci was observed in Tlr2^+/+ but not Tlr2^–/– mice (Figure 5, A and B). The expansion of this population in Tlr2^+/+ mice correlated with an increase in numbers of lymphocytes in cytospin preparations and CD4⁺ T cells by flow cytometry in nasal lavages (Figure 5, C and D).

Figure 5

Colonization with strain P1121 led to mucosal and systemic CD4⁺ T cell responses, which were attenuated in the absence of TLR2 signaling. Mice were colonized with strain P1121 (or sham colonized for naive animals) for 6 weeks, and T cell responses to P1121 were analyzed. (A) Systemic Th17 response in P1121-colonized WT but not Tlr2^–/– mice. Splenocytes were stimulated ex vivo with heat-killed P1121 (MOI 50:1), and P1121-specific Th17 response was evaluated by quantification of CD45⁺– and CD4⁺–double-positive events through IL-17A intracellular cytokine staining analysis. Representative experiment showing responses in naive and previously colonized WT mice (upper panels) or Tlr2^–/– mice (lower panels), with the numbers in the upper left-hand corner indicating the frequencies of IL-17A–producing CD4⁺ T cells. (B) Combined results of IL-17A intracellular cytokine staining of splenocytes in response to stimulation with heat-killed P1121 (hk-P1121), medium (negative control), and a combination of PMA (6 nM) and ionomycin (5 nM) (positive control [Pos]). (C) Mucosal T cell responses are blunted in Tlr2^–/– mice. WT (black bars) or Tlr2^–/– mice (white bars) were challenged with P1121 (primary) or rechallenged with P1121 for 1 day 6 weeks after precolonization (secondary). The number of CD4⁺ T cells at the mucosal surface was quantified by differential cell quantification of lymphocytes in cytospin preparations; n = 5 per group. (D) Representative experiment showing flow cytometric quantification of mucosal CD4⁺ T cells. Numbers of CD45⁺– and CD4⁺–double-positive events in pooled nasal washes are shown in the upper left-hand corners. n = 5 per group. *P < 0.05; **P < 0.01.

When compared with primary colonization, there was a more rapid clearance of pneumococci during secondary challenge (P = 0.003 at day 5 after inoculation; Figure 6A). This more rapid clearance was associated with consistently higher numbers of monocyte/macrophages during the observation period, although these differences did not achieve statistical significance until day 5 after inoculation (P = 0.03; Figure 6C). Associated with the increased recruitment of monocytes/macrophages following secondary challenge, there was an increase in transcription of the CCL2/monocyte chemotactic protein-1 (MCP-1) in the nasal epithelium (P = 0.01; Figure 6D). Moreover, the increased expression of MCP-1 during secondary challenge was not observed in mice previously depleted of CD4⁺ T cells or lacking TLR2. Secondary challenge was also associated with an earlier and more robust neutrophil response (P = 0.0006 at day 1 after inoculation; Figure 6B). Depletion of CD4⁺ but not CD8⁺ T cells at the time of secondary challenge completely abolished the enhanced early clearance (P = 0.001; Figure 7A) as well as the increased monocyte/macrophage (P = 0.001; Figure 7C) and neutrophil (P = 0.006; Figure 7B) recruitment associated with reinfection. Similar to depletion of CD4⁺ T cells, depletion of IL-17A prior to secondary challenge eliminated the increased clearance observed upon reinfection (P = 0.02; Figure 7A). Depletion of IL-17A, furthermore, blocked recruitment of both neutrophils (P = 0.02; Figure 7B) and monocyte/macrophages (P = 0.008; Figure 7C) into the nasal spaces. Previously, we had reported that the depletion of Ly6G-expressing neutrophil-like cells using the mAb RB6-8C5 has a minimal impact on colonization during primary challenge (24). During secondary challenge, however, treatment with RB6-8C5, which was effective at depleting neutrophils without affecting monocyte/macrophages, inhibited early clearance (P = 0.03; Figure 7A).

Figure 6

Accelerated clearance in the secondary pneumococcal challenge is associated with enhanced cellular responses. Comparisons of the time course of (A) the density of P1121 colonization, (B) neutrophil recruitment, and (C) monocyte/macrophage recruitment during primary (closed circles) and secondary (open circles) challenge. n = 5–20 mice per group per time point. Values represent mean ± SEM. (D) Quantitative real-time RT-PCR showing increased transcription of chemokine MCP-1 in the colonized epithelium of the nasopharynx. WT (black bars) or Tlr2^–/– (white bars) mice were challenged with P1121 (Primary) or rechallenged with P1121 for one day 6 weeks after precolonization (Secondary). WT mice were also treated with GK1.5 antibodies to deplete CD4⁺ T cells (gray bar) prior to rechallenge. n = 4 mice per group. *P < 0.05, **P < 0.01; ***P < 0.001.

Figure 7

Host factors required for accelerated pneumococcal clearance during secondary challenge. Nasal lavages were assessed at 1 day following secondary challenge for (A) density of P1121 colonization, (B) neutrophil recruitment, and (C) monocyte/macrophage recruitment. Prior to rechallenge, mice were treated with RB6-8C5 mAbs to deplete Ly6G-positive neutrophil-like cells, anti–IL-17A to neutralize IL-17A, 2.43 antibodies to deplete CD8⁺ T cells, or GK1.5 antibodies to deplete CD4⁺ T cells and compared with untreated controls. Values represent individual animals, with the mean shown by a horizontal bar. *P < 0.05; **P < 0.01; ***P < 0.001.

Taken together, these data suggest that primary challenge generates CD4⁺ T cell memory, resulting in enhanced Th17-mediated recruitment of cellular effectors, including both monocyte/macrophages and neutrophils. In contrast with primary colonization, neutrophils recruited in response to secondary challenge contribute to early clearance.

We propose a model for the clearance of S. pneumoniae from the mucosal surface of the nasopharynx with many of the features of a delayed-type hypersensitivity response. In particular, our findings demonstrate that bacteria induce CD4⁺ T cell–mediated cellular immunity that is necessary to sustain the local recruitment of nonresident monocyte/macrophages into the upper airway lumen. This infiltration of monocyte/macrophages is prolonged, lasting for several weeks until the organism is no longer detectable, although these cells never appear in large numbers in upper airway lavages. These observations could account for the typical duration and transient nature of episodes of pneumococcal colonization. Several lines of evidence support the conclusion that monocyte/macrophages recruited into the airway lumen act as key effector cells to clear colonizing pneumococci: (a) the time course of recruitment parallels clearance, (b) pharmacologic depletion of these cells impedes clearance, (c) pneumococci are found engulfed by these cells during the time period when the density of colonizing bacteria is declining, and (d) neutralization of each of the host factors needed for monocyte/macrophage recruitment, including TLR2, CD4⁺ T cells, and IL-17A, results in diminished clearance. Macrophages have been shown to be important in host defense when infection spreads to normally sterile sites, such as the lung, where pneumococci are targeted by alveolar macrophages (25, 26). In his classic monograph on pneumococcal pneumonia, Heffron describes “the macrophage reaction” and that “associated with the increasing proportion of macrophages in the exudates in late resolution there was a concurrent decrease in the number of pneumococci” (27). It has recently been demonstrated that in the course of pneumococcal pneumonia, there is a brisk turnover of resident alveolar macrophages and replacement with newly recruited monocytes (28, 29). The present study demonstrates that monocyte/macrophages recruited into the lumen of the upper airway in response to colonization might serve an analogous function in clearing pneumococci in their commensal state.

Several factors were identified as contributing to the mobilization of monocyte/macrophages into the upper airway lumen. There was a requirement for bacteria on the mucosal surface, since numbers of monocyte/macrophages were consistently low in precolonized mice. During the first 3 days of colonization, there is a TLR2-independent influx of monocyte/macrophages and neutrophils. Epithelial surfaces initiate proinflammatory responses to colonizing microbes that result in the elaboration of chemokines and cytokines. For example, 2 epithelial cell–originated antimicrobial peptides, S100A8 and S100A9, have recently been shown to possess chemotactic properties that are required for efficient recruitment of phagocytes, such as macrophages and neutrophils, into the lung in a streptococcal pneumonia mouse model (30). Additional data in this report demonstrate a critical role for TLR2-mediated signaling and CD4⁺ T cells in maintaining monocyte/macrophage recruitment during later stages of colonization. TLR2 signaling may be required for efficient antigen presentation and initiation of an effective T cell response. This could explain why the effect of TLR2 is observed at day 21 after inoculation but not earlier, as this is prior to the development of adaptive immunity. Helper T cells transmit signals to monocyte/macrophages to promote killing of intracellular organisms. For typical extracellular pathogens, such as S. pneumoniae, the importance and identity of the molecular signals provided by CD4⁺ T cells to stimulate the recruitment and activity of the monocyte/macrophage effectors have not been established. The predominant IgG isotypes generated against the major surface protein antigen of strain P1121 are IgG2b and IgG3, subtypes typically generated in a Th1-biased immune response (18). We were unable, however, to demonstrate any contribution of the Th1 cytokine IFN-γ, since mice lacking its receptor show normal clearance of colonization (data not shown). Th17 cells, a subset of CD4⁺ T cells, are characterized by the ability to generate IL-17A, a cytokine responsible for governing both neutrophil- and macrophage-characterized inflammation, especially in chronic immune disorders or infections of the respiratory tract (31–33). Early pneumococcal colonization stimulates transcription of IL-6 in the nasal epithelium, a response that may contribute to the induction of IL-17–expressing cells (34). It has been suggested that IL-17 is produced systemically while it exerts proinflammatory effects locally through regulation of epithelial cells that are able to express a broad spectrum of chemokines recruiting immune effector cells, including neutrophils and macrophage precursor cells (35–37). In this study, the expression of MCP-1, a chemokine that can recruit monocytes in a Th17-dependent manner, was increased during secondary pneumococcal challenge (31). Endogenous production of IL-17, moreover, has been proposed as a recruitment factor with direct chemotactic effects on blood monocytes (37). Colonization was shown to induce a population of IL-17–expressing CD4⁺ T cells reactive with pneumococci. Moreover, our study indicates that an IL-17–expressing subpopulation of CD4⁺ T cells mediates the recruitment of luminal monocyte/macrophages (primary and secondary challenge) and neutrophils (secondary challenge). Our study, therefore, expands on the reports by Malley and coworkers of a Th17 cell response to pneumococcal antigens, to demonstrate that during the natural course of pneumococcal colonization, these cells are critically required for the recruitment of phagocytes that leads to the clearance of colonization in both naive and immune hosts (38). Our findings about pneumococcal colonization, furthermore, are consistent with reports demonstrating that Th17 responses are critical for immunity to extracellular bacterial pathogens during infection (39).

A further question is how monocyte/macrophages recruited into the airway lumen recognize bacterial targets. Previous reports suggest that their function in clearing bacteria from the mucosal surface is not likely to require the presence of specific antibody coating of target organisms during primary colonization (11, 14). Complement components could act as opsonins to facilitate phagocytosis (40). It has also been suggested that early clearance from the lung by alveolar macrophages may proceed through a complement-independent mechanism (41). Other molecules found in airway secretions, such as C-reactive protein, are capable of opsonizing pneumococci (42–44). In addition, surface components of monocyte/macrophages, including the C-type lectin SIGNR1, pulmonary surfactant proteins of the collectin family, and the scavenger receptor MARCO, that promote direct recognition and phagocytosis of pneumococci have been described (45–47).

In naive mice, the acquisition of bacteria in the upper airway induces an influx of neutrophils. This acute inflammatory response, however, is insufficient to clear the organism from the mucosal surface (24). A subsequent process that involves CD4⁺ T cell–dependent recruitment of monocyte/macrophages is required to target persisting bacteria following the resolution of the acute inflammatory response during initial colonization. The situation in previously exposed mice, which acquire CD4⁺ T cell memory, appears to be distinct, as there is an earlier IL-17A–dependent influx of professional phagocytes. After secondary challenge, the neutrophil influx, in particular, occurs more quickly and is of a greater magnitude. Importantly, in contrast with the response in naive mice, the neutrophil response during secondary challenge contributes to early bacterial clearance. This finding in secondary challenge might be attributable to opsonizing antibody in immune mice that facilitates phagocytosis when neutrophils are present. In primary challenge, on the other hand, the influx of neutrophils occurs before neutralizing antibody is generated.

In summary, our study provides insight into the events leading to clearance of pneumococcal carriage and offers a new paradigm for understanding how colonizing microbes are targeted by cellular immunity.

We thank the Cell Morphology Core of the Gene Therapy Program, the Morphology Core of the Center for the Molecular Studies of Liver and Digestive Diseases (P30 DK50306), and the Biomedical Imaging Facility at the University of Pennsylvania for technical support. We thank Elena Lysenko and Xiaobo Wang for technical assistance. This work was funded by grants from the US Public Health Service (AI44231 and AI38446 to J.N. Weiser).

Address correspondence to: Jeffrey N. Weiser, University of Pennsylvania School of Medicine, 402A Johnson Pavilion, 3610 Hamilton Walk, Philadelphia, Pennsylvania 19104-6076, USA. Phone: (215) 573-3511; Fax: (215) 573-4856; E-mail: weiser@mail.med.upenn.edu.

Conflict of interest: The authors have declared that no conflict of interest exists.

Nonstandard abbreviations used: APC, allophycocyanin; Cl₂MDP, clodronate; LAMP-1, lysosomal marker lysosome-associated membrane protein-1; MCP-1, monocyte chemotactic protein-1; MOMA-2, monocyte/macrophage marker; PerCP, peridinin chlorophyll protein.

Reference information: J. Clin. Invest.119:1899–1909 (2009). doi:10.1172/JCI36731

1. Cutts, F.T., et al. 2005. Efficacy of nine-valent pneumococcal conjugate vaccine against pneumonia and invasive pneumococcal disease in The Gambia: randomised, double-blind, placebo-controlled trial. Lancet. 365:1139-1146.
   View this article via: CrossRef PubMed Google Scholar
2. Kadioglu, A., Weiser, J.N., Paton, J.C., Andrew, P.W. 2008. The role of Streptococcus pneumoniae virulence factors in host respiratory colonization and disease. Nat. Rev. Microbiol. 6:288-301.
   View this article via: CrossRef PubMed Google Scholar
3. Austrian, R. 1986. Some aspects of the pneumococcal carrier state. J. Antimicrob. Chemother. 18:35-45.
   View this article via: CrossRef PubMed Google Scholar
4. Bogaert, D., De Groot, R., Hermans, P.W. 2004. Streptococcus pneumoniae colonisation: the key to pneumococcal disease. Lancet. Infect. Dis. 4:144-154.
   View this article via: CrossRef PubMed Google Scholar
5. Syrjanen, R.K., Kilpi, T.M., Kaijalainen, T.H., Herva, E.E., Takala, A.K. 2001. Nasopharyngeal carriage of Streptococcus pneumoniae in Finnish children younger than 2 years old. J. Infect. Dis. 184:451-459.
   View this article via: CrossRef PubMed Google Scholar
6. McCool, T.L., Cate, T.R., Moy, G., Weiser, J.N. 2002. The immune response to pneumococcal proteins during experimental human carriage. J. Exp. Med. 195:359-365.
   View this article via: CrossRef PubMed Google Scholar
7. Goldblatt, D., et al. 2005. Antibody responses to nasopharyngeal carriage of Streptococcus pneumoniae in adults: a longitudinal household study. J. Infect. Dis. 192:387-393.
   View this article via: CrossRef PubMed Google Scholar
8. MacLeod, C.M., Hodges, R.M., Heidelberger, M., Bernhard, W.G. 1945. Prevention of pneumococcal infection by immunization with capsular polysaccharides of Streptococcus pneumoniae. J. Exp. Med. 82:445-465.
   View this article via: CrossRef Google Scholar
9. Dagan, R., et al. 2005. Serum serotype-specific pneumococcal anticapsular immunoglobulin g concentrations after immunization with a 9-valent conjugate pneumococcal vaccine correlate with nasopharyngeal acquisition of pneumococcus. J. Infect. Dis. 192:367-376.
   View this article via: CrossRef PubMed Google Scholar
10. Mbelle, N., et al. 1999. Immunogenicity and impact on nasopharyngeal carriage of a nonavalent pneumococcal conjugate vaccine. J. Infect. Dis. 180:1171-1176.
    View this article via: CrossRef PubMed Google Scholar
11. McCool, T.L., Weiser, J.N. 2004. Limited role of antibody in clearance of Streptococcus pneumoniae in a murine model of colonization. Infect. Immun. 72:5807-5813.
    View this article via: CrossRef PubMed Google Scholar
12. Lipsitch, M., et al. 2005. Are anticapsular antibodies the primary mechanism of protection against invasive pneumococcal disease? PLoS Med. 2:e15.
    View this article via: PubMed Google Scholar
13. Malley, R., et al. 2004. Multiserotype protection of mice against pneumococcal colonization of the nasopharynx and middle ear by killed nonencapsulated cells given intranasally with a nontoxic adjuvant. Infect. Immun. 72:4290-4292.
    View this article via: CrossRef PubMed Google Scholar
14. Malley, R., et al. 2005. CD4+ T cells mediate antibody-independent acquired immunity to pneumococcal colonization. Proc. Natl. Acad. Sci. U. S. A. 102:4848-4853.
    View this article via: CrossRef PubMed Google Scholar
15. Malley, R., et al. 2006. Antibody-independent, interleukin-17A-mediated, cross-serotype immunity to pneumococci in mice immunized intranasally with the cell wall polysaccharide. Infect. Immun. 74:2187-2195.
    View this article via: CrossRef PubMed Google Scholar
16. Trzcinski, K., et al. 2008. Protection against nasopharyngeal colonization by Streptococcus pneumoniae is mediated by antigen-specific CD4+ T cells. Infect. Immun. 76:2678-2684.
    View this article via: CrossRef PubMed Google Scholar
17. Madhi, S.A., et al. 2007. Long-term effect of pneumococcal conjugate vaccine on nasopharyngeal colonization by Streptococcus pneumoniae — and associated interactions with Staphylococcus aureus and Haemophilus influenzae colonization--in HIV-Infected and HIV-uninfected children. J. Infect. Dis. 196:1662-1666.
    View this article via: CrossRef PubMed Google Scholar
18. van Rossum, A.M., Lysenko, E.S., Weiser, J.N. 2005. Host and bacterial factors contributing to the clearance of colonization by Streptococcus pneumoniae in a murine model. Infect. Immun. 73:7718-7726.
    View this article via: CrossRef PubMed Google Scholar
19. Akira, S. 2006. TLR signaling. Curr. Top. Microbiol. Immunol. 311:1-16.
    View this article via: CrossRef PubMed Google Scholar
20. Kabelitz, D. 2007. Expression and function of Toll-like receptors in T lymphocytes. Curr. Opin. Immunol. 19:39-45.
    View this article via: CrossRef PubMed Google Scholar
21. Malley, R., et al. 2003. Recognition of pneumolysin by Toll-like receptor 4 confers resistance to pneumococcal infection. Proc. Natl. Acad. Sci. U. S. A. 100:1966-1971.
    View this article via: CrossRef PubMed Google Scholar
22. Kadioglu, A., Coward, W., Colston, M.J., Hewitt, C.R., Andrew, P.W. 2004. CD4-T-lymphocyte interactions with pneumolysin and pneumococci suggest a crucial protective role in the host response to pneumococcal infection. Infect. Immun. 72:2689-2697.
    View this article via: CrossRef PubMed Google Scholar
23. Lu, Y.J., et al. 2008. Interleukin-17A mediates acquired immunity to pneumococcal colonization. PLoS Pathog. 4:e1000159.
    View this article via: PubMed Google Scholar
24. Matthias, K.A., Roche, A.M., Standish, A.J., Shchepetov, M., Weiser, J.N. 2008. Neutrophil-toxin interactions promote antigen delivery and mucosal clearance of Streptococcus pneumoniae. J. Immunol. 180:6246-6254.
    View this article via: PubMed Google Scholar
25. Bergeron, Y., et al. 1998. Cytokine kinetics and other host factors in response to pneumococcal pulmonary infection in mice. Infect. Immun. 66:912-922.
    View this article via: PubMed Google Scholar
26. Sun, K., Metzger, D.W. 2008. Inhibition of pulmonary antibacterial defense by interferon-gamma during recovery from influenza infection. Nat. Med. 14:558-564.
    View this article via: CrossRef PubMed Google Scholar
27. Heffron, R. 1979. Pneumonia, with special reference to pneumococcus lobar pneumonia. Harvard University Press. Cambridge, Massachusetts, USA. 1086 pp.
28. Taut, K., et al. 2008. Macrophage turnover kinetics in the lungs of mice infected with Streptococcus pneumoniae. Am. J. Respir. Cell Mol. Biol. 38:105-113.
    View this article via: PubMed Google Scholar
29. Maus, U., et al. 2001. Monocytes recruited into the alveolar air space of mice show a monocytic phenotype but upregulate CD14. Am. J. Physiol. Lung Cell Mol. Physiol. 280:L58-L68.
    View this article via: PubMed Google Scholar
30. Raquil, M.A., Anceriz, N., Rouleau, P., Tessier, P.A. 2008. Blockade of antimicrobial proteins S100A8 and S100A9 inhibits phagocyte migration to the alveoli in streptococcal pneumonia. J. Immunol. 180:3366-3374.
    View this article via: PubMed Google Scholar
31. Park, H., et al. 2005. A distinct lineage of CD4 T cells regulates tissue inflammation by producing interleukin 17. Nat. Immunol. 6:1133-1141.
    View this article via: CrossRef PubMed Google Scholar
32. Harrington, L.E., et al. 2005. Interleukin 17-producing CD4+ effector T cells develop via a lineage distinct from the T helper type 1 and 2 lineages. Nat. Immunol. 6:1123-1132.
    View this article via: CrossRef PubMed Google Scholar
33. Bettelli, E., et al. 2006. Reciprocal developmental pathways for the generation of pathogenic effector TH17 and regulatory T cells. Nature. 441:235-238.
    View this article via: CrossRef PubMed Google Scholar
34. Beisswenger, C., Lysenko, E.S., Weiser, J.N. 2009. Early bacterial colonization induces toll-like receptor–dependent transforming growth factor beta signaling in the epithelium. Infect. Immun. 77:2212-2220.
    View this article via: CrossRef PubMed Google Scholar
35. Miyamoto, M., et al. 2003. Endogenous IL-17 as a mediator of neutrophil recruitment caused by endotoxin exposure in mouse airways. J. Immunol. 170:4665-4672.
    View this article via: PubMed Google Scholar
36. Kolls, J.K., Linden, A. 2004. Interleukin-17 family members and inflammation. Immunity. 21:467-476.
    View this article via: CrossRef PubMed Google Scholar
37. Sergejeva, S., Ivanov, S., Lotvall, J., Linden, A. 2005. Interleukin-17 as a recruitment and survival factor for airway macrophages in allergic airway inflammation. Am. J. Respir. Cell Mol. Biol. 33:248-253.
    View this article via: CrossRef PubMed Google Scholar
38. Basset, A., et al. 2007. Antibody-independent, CD4+ T-cell-dependent protection against pneumococcal colonization elicited by intranasal immunization with purified pneumococcal proteins. Infect. Immun. 75:5460-5464.
    View this article via: CrossRef PubMed Google Scholar
39. Aujla, S.J., Dubin, P.J., Kolls, J.K. 2007. Th17 cells and mucosal host defense. Semin. Immunol. 19:377-382.
    View this article via: CrossRef PubMed Google Scholar
40. van Lookeren Campagne, M., Wiesmann, C., Brown, E.J. 2007. Macrophage complement receptors and pathogen clearance. Cell. Microbiol. 9:2095-2102.
    View this article via: CrossRef PubMed Google Scholar
41. Rehm, S.R., Coonrod, J.D. 1982. Early clearance of pneumococci from the lungs of decomplemented rats. Infect. Immun. 36:24-29.
    View this article via: PubMed Google Scholar
42. Gould, J.M., Weiser, J.N. 2001. Expression of C-reactive protein in the human respiratory tract. Infect. Immun. 69:1747-1754.
    View this article via: CrossRef PubMed Google Scholar
43. Szalai, A.J., Briles, D.E., Volanakis, J.E. 1995. Human C-reactive protein is protective against fatal Streptococcus pneumoniae infection in transgenic mice. J. Immunol. 155:2557-2563.
    View this article via: PubMed Google Scholar
44. Gould, J.M., Weiser, J.N. 2002. The inhibitory effect of C-reactive protein on bacterial phosphorylcholine platelet-activating factor receptor-mediated adherence is blocked by surfactant. J. Infect. Dis. 186:361-371.
    View this article via: CrossRef PubMed Google Scholar
45. Kuronuma, K., et al. 2004. Pulmonary surfactant protein A augments the phagocytosis of Streptococcus pneumoniae by alveolar macrophages through a casein kinase 2-dependent increase of cell surface localization of scavenger receptor A. J. Biol. Chem. 279:21421-21430.
    View this article via: CrossRef PubMed Google Scholar
46. Koppel, E.A., Litjens, M., van den Berg, V.C., van Kooyk, Y., Geijtenbeek, T.B. 2008. Interaction of SIGNR1 expressed by marginal zone macrophages with marginal zone B cells is essential to early IgM responses against Streptococcus pneumoniae. Mol. Immunol. 45:2881-2887.
    View this article via: CrossRef PubMed Google Scholar
47. Arredouani, M., et al. 2004. The scavenger receptor MARCO is required for lung defense against pneumococcal pneumonia and inhaled particles. J. Exp. Med. 200:267-272.
    View this article via: CrossRef PubMed Google Scholar
48. Takeuchi, O., et al. 1999. Differential roles of TLR2 and TLR4 in recognition of gram-negative and gram-positive bacterial cell wall components. Immunity. 11:443-451.
    View this article via: CrossRef PubMed Google Scholar
49. Weiser, J.N., Austrian, R., Sreenivasan, P.K., Masure, H.R. 1994. Phase variation in pneumococcal opacity: relationship between colonial morphology and nasopharyngeal colonization. Infect. Immun. 62:2582-2589.
    View this article via: PubMed Google Scholar
50. Roche, A.M., King, S.J., Weiser, J.N. 2007. Live attenuated Streptococcus pneumoniae strains induce serotype-independent mucosal and systemic protection in mice. Infect. Immun. 75:2469-2475.
    View this article via: CrossRef PubMed Google Scholar
51. Cotter, M.J., Norman, K.E., Hellewell, P.G., Ridger, V.C. 2001. A novel method for isolation of neutrophils from murine blood using negative immunomagnetic separation. Am. J. Pathol. 159:473-481.
    View this article via: PubMed Google Scholar
52. Van Rooijen, N. 1989. The liposome-mediated macrophage ‘suicide’ technique. J. Immunol. Methods. 124:1-6.
    View this article via: CrossRef PubMed Google Scholar
53. Pan, Z.K., Ikonomidis, G., Lazenby, A., Pardoll, D., Paterson, Y. 1995. A recombinant Listeria monocytogenes vaccine expressing a model tumour antigen protects mice against lethal tumour cell challenge and causes regression of established tumours. Nat. Med. 1:471-477.
    View this article via: CrossRef PubMed Google Scholar
54. Lysenko, E.S., Ratner, A.J., Nelson, A.L., Weiser, J.N. 2005. The role of innate immune responses in the outcome of interspecies competition for colonization of mucosal surfaces. PLoS Pathog. 1:e1.
    View this article via: PubMed Google Scholar
55. [No authors listed]. 2008. Guide to performing relative quantitation of gene expression using real-time quantitative PCR. Applied Biosystems Inc. Foster City, California, USA. http://www3.appliedbiosystems.com/cms/groups/mcb_support/documents/generaldocuments/cms_042380.pdf