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. 2015 Jun 24;11(6):e1005004.
doi: 10.1371/journal.ppat.1005004. eCollection 2015 Jun.

Clearance of Pneumococcal Colonization in Infants Is Delayed through Altered Macrophage Trafficking

Steven J Siegel  1 , Edwin Tamashiro  2 , Jeffrey N Weiser  3
Affiliations

Clearance of Pneumococcal Colonization in Infants Is Delayed through Altered Macrophage Trafficking

Steven J Siegel et al. PLoS Pathog. .

Abstract

Infections are a common cause of infant mortality worldwide, especially due to Streptococcus pneumoniae. Colonization is the prerequisite to invasive pneumococcal disease, and is particularly frequent and prolonged in children, though the mechanisms underlying this susceptibility are unknown. We find that infant mice exhibit prolonged pneumococcal carriage, and are delayed in recruiting macrophages, the effector cells of clearance, into the nasopharyngeal lumen. This lack of macrophage recruitment is paralleled by a failure to upregulate chemokine (C-C) motif ligand 2 (Ccl2 or Mcp-1), a macrophage chemoattractant that is required in adult mice to promote clearance. Baseline expression of Ccl2 and the related chemokine Ccl7 is higher in the infant compared to the adult upper respiratory tract, and this effect requires the infant microbiota. These results demonstrate that signals governing macrophage recruitment are altered at baseline in infant mice, which prevents the development of appropriate innate cell infiltration in response to pneumococcal colonization, delaying clearance of pneumococcal carriage.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Pneumococcal carriage is prolonged in infant mice.
(A) Adult (6 weeks old) and infant (7d old) mice were inoculated with pneumococcal strain P1121, and at the indicated number of days postinoculation (dpi), mice were sacrificed and nasal lavages obtained and plated to determine the load of colonizing pneumococci. (B) Adult and infant mice were inoculated with pneumococcal strain TIGR4 (type 4). Mice were sacrificed at 21 dpi and nasal lavages obtained and plated to determine the load of colonizing pneumococci. (C) Mice were inoculated with strain P1121 (type 23F) at different ages, ranging from 7 to 42 days. At 21 dpi, mice were sacrificed and nasal lavages obtained and plated to measure bacterial density. Points in (A) represent mean +/- SEM, with 5–18 mice per group. Horizontal lines indicate median values. Dotted lines indicate limit of detection. n.s. = not significant, ** = p < 0.01, *** = p < 0.001.
Fig 2
Fig 2. Infant mice are impaired in macrophage recruitment during colonization.
(A-C) Adult (6 week old) and infant (7d old) mice were inoculated with strain P1121 for the indicated number of days. Nasal lavages were obtained and fixed and stained for flow cytometry to identify macrophages (F4/80+, CD11b-) and neutrophils (CD11b+, Ly6G+). CD11b surface expression was measured on myeloid cells and displayed as median fluorescence intensity. Samples represent at least 10 mice per timepoint. (D) Serum was obtained from adult and infant mice colonized with pneumococci for 21 days, or mock-colonized. Samples were analyzed by ELISA for the presence of antibodies specific to strain P1121. (E) Infant mice of the indicated genotype were colonized at 7d of age. Mice were sacrificed at the indicated timepoints and nasal lavages obtained and plated to measure bacterial load. Data are represented as mean +/- SEM. n.s. = not significant, * = p < 0.05, ** = p < 0.01.
Fig 3
Fig 3. Infant mice do not form a gradient of CCL2 expression during colonization.
(A) Adult (42d old) and infant (7d old) mice were inoculated with PBS (mock) or pneumococci. At 3d post-inoculation, nasal lavages were obtained with RLT lysis buffer, and RNA was isolated and reverse transcribed to cDNA. qRT-PCR was performed to measure relative expression of Ccl2. (B) Serum was obtained from adult and infant mice, and ELISA used to measure CCL2 levels. (C-D) Peritoneal macrophages from adult and infant mice were lysed with RLT buffer and RNA isolated. qRT-PCR was used to measure relative expression of Ccl2 (C) and Ccr2 (D). In (C), cultured macrophages were incubated overnight with PBS (Mock) or heat-killed bacterial lysates (Stim) prior to lysis. (E-H) Uncolonized adult and infant mice were sacrificed, and nasal lavages obtained with RLT lysis buffer. RNA was isolated and reverse-transcribed into cDNA. qRT-PCR was used to measure relative expression of Ccl7, (E) Il6, (F) Cxcl1, (G) and Cxcl2 (H). Data are represented as mean +/- SEM. n.s., not significant. * = p < 0.05, ** = p < 0.01, *** = p < 0.001.
Fig 4
Fig 4. CCL2 overexpression increases macrophage recruitment and pneumococcal clearance.
(A-C) Mice were inoculated with a control (GFP-expressing) or a CCL2-expressing AAV5 vector at 4d of age, followed by pneumococcal colonization at 7d old. Seven and 21 days later, mice were sacrificed and nasal lavages were obtained with RLT lysis buffer. (A) RNA was isolated and cDNA reverse-transcribed, followed by qRT-PCR to measure the relative expression of Ccl2 in the upper respiratory tract, using primers Ccl2ORF-F and Ccl2ORF-R. ELISA was used to measure CCL2 protein levels in nasal lavage (B) and serum (C) at 7 and 21 dpi. (D) Mice were inoculated with a control (GFP-expressing) or a CCL2-expressing AAV5 vector at 4d of age, followed by pneumococcal colonization at 7d of age. Seven days later, mice were sacrificed, nasal lavages obtained and flow cytometry used to measure macrophage recruitment. (E) At 21d post-inoculation, nasal lavages were obtained and plated to measure the pneumococcal load in the nasopharynx. Data are represented as mean +/- SEM. n.s., not significant. * = p < 0.05, *** = p < 0.001. Dotted line, limit of detection.
Fig 5
Fig 5. Depleting the microbiota limits infant Ccl2 expression and accelerates pneumococcal clearance.
(A) Breeding pairs were placed on sterile filtered tap water or water containing 5 antibiotics (Abx) for at least 5 days prior to giving birth, and continued until the infant mice were 10d old. Mice were sacrificed, and nasal lavages obtained with PBS. DNA was isolated from lavage fluid, and qPCR used to measure the number of copies of 16S bacterial ribosomal DNA. (B-C) Adult mice were maintained on sterile filtered tap water or water containing 5 antibiotics for at least 2 weeks. Breeding pairs were provided water as above. Mice were sacrificed, and nasal lavages obtained with RLT lysis buffer. RNA was isolated and reverse-transcribed into cDNA, and qRT-PCR used to measure relative expression of Ccl2 (B) and Ccl7 (C) in the nasopharynx. (D) Infant mice were exposed to tap water or antibiotics as in the previously described protocol. Treatments were continued until infant mice were 6d old, then all mice were given tap water to drink. 24 hrs later, mice were inoculated with pneumococci. At 7 dpi, mice were sacrificed and nasal lavages fixed and stained for flow cytometry to measure the number of macrophages present in the airway lumen. (E-F) Infant mice were treated as in (D). At 14d (E) and 7d (F) post-inoculation, mice were sacrificed. Nasal lavages were obtained and plated on media containing neomycin and catalase to measure pneumococcal density in the URT. (G) Ccl2 expression was measured in the nasopharynx of tap- or antibiotic-water exposed conventionally-reared (Conv), or germ-free infant mice. Data are represented as mean +/- SEM. Horizontal lines indicate median values. n.s., not significant. * = p < 0.05, ** = p < 0.01, *** = p < 0.001.
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