Infant mice are impaired in macrophage recruitment during colonization.

<p>(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.</p

CCL2 overexpression increases macrophage recruitment and pneumococcal clearance.

<p>(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 <i>Ccl2</i> in the upper respiratory tract, using primers <i>Ccl2</i>ORF-F and <i>Ccl2</i>ORF-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.</p

Infant mice do not form a gradient of CCL2 expression during colonization.

<p>(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 <i>Ccl2</i>. (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 <i>Ccl2</i> (C) and <i>Ccr2</i> (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 <i>Ccl7</i>, (E) <i>Il6</i>, (F) <i>Cxcl1</i>, (G) and <i>Cxcl2</i> (H). Data are represented as mean +/- SEM. n.s., not significant. * = p < 0.05, ** = p < 0.01, *** = p < 0.001.</p

The mGluR5-receptor antagonist MPEP attenuates elevated repetitive behaviors in mice exposed to valproic acid (VPA) <i>in utero.</i>

<p>(<b>A</b>) Repetitive self-grooming was measured over 10 minutes in mice exposed to prenatal saline (SAL) or VPA. (<b>B</b>) Repetitive marble burying behavior was measured for both groups after a 30 minute testing session. Across both assays, VPA-exposed mice demonstrated elevated stereotyped, repetitive behaviors that were significantly reduced by MPEP. Figures show mean ± S.E.M., (*<i>p</i><0.05, **<i>p</i><0.01).</p

Pneumococcal carriage is prolonged in infant mice.

<p>(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.</p

The effect of MPEP was assessed on anxiety-like behavior in VPA- and SAL-exposed mice using an open-field paradigm.

<p>Consistent with elevated anxiety, VPA-exposed mice demonstrated significantly fewer (<b>A</b>) center entries and qualitatively reduced (<b>B</b>) center time. Across groups, there was no effect of MPEP on either of these measures. Figures show mean ± S.E.M., (#<i>p</i><0.1, *<i>p</i><0.05).</p

TLR2 Signaling Decreases Transmission of <i>Streptococcus pneumoniae</i> by Limiting Bacterial Shedding in an Infant Mouse Influenza A Co-infection Model

<div><p>While the importance of transmission of pathogens is widely accepted, there is currently little mechanistic understanding of this process. Nasal carriage of <i>Streptococcus pneumoniae</i> (the pneumococcus) is common in humans, especially in early childhood, and is a prerequisite for the development of disease and transmission among hosts. In this study, we adapted an infant mouse model to elucidate host determinants of transmission of <i>S. pneumoniae</i> from inoculated index mice to uninfected contact mice. In the context of co-infection with influenza A virus, the pneumococcus was transmitted among wildtype littermates, with approximately half of the contact mice acquiring colonization. Mice deficient for TLR2 were colonized to a similar density but transmitted <i>S. pneumoniae</i> more efficiently (100% transmission) than wildtype animals and showed decreased expression of interferon α and higher viral titers. The greater viral burden in <i>tlr2^−/−</i> mice correlated with heightened inflammation, and was responsible for an increase in bacterial shedding from the mouse nose. The role of TLR2 signaling was confirmed by intranasal treatment of wildtype mice with the agonist Pam3Cys, which decreased inflammation and reduced bacterial shedding and transmission. Taken together, these results suggest that the innate immune response to influenza virus promotes bacterial shedding, allowing the bacteria to transit from host to host. These findings provide insight into the role of host factors in the increased pneumococcal carriage rates seen during flu season and contribute to our overall understanding of pathogen transmission.</p></div

Influenza infection results in neutrophil influx to the nasopharynx.

<p>Wildtype mice were infected as index mice with the listed agents. On day 14, mice were euthanized, and nasal lavage was performed with PBS. <b>A.</b> A 100 µl sample of each lavage was analyzed by flow cytometry to detect total numbers of neutrophils (Ly6G⁺, CD11b⁺). Each dot represents the percentage of total events that were neutrophils per mouse. Statistical significance was assessed using the Kruskal-Wallis test followed by Dunn's post-test, and * indicates <i>p</i><0.05, while ** denotes <i>p</i><0.01. <b>B.</b> Immunofluorescence image (representative). Ten microliter samples of nasal lavage fluid were spotted onto a glass slide, fixed, and stained with DAPI (blue) and antibodies against Ly6B (green) and type 23F pneumococcal capsule polysaccharide (red).</p

<i>S. pneumoniae</i> is transmitted between hosts in an infant mouse influenza co-infection model.

<p><b>A.</b> Schematic of inoculation schedule. Listed agents were administered intranasally to unanesthetized mice in a volume of 3 µl PBS and pups were returned to their mothers. Numbered days refer to age of mice, which were euthanized on day 14. Nasal lavage was performed with 200 µl PBS, which was then serially diluted and plated on selective agar to obtain colony counts. <b>B.</b> Pups were infected and sacrificed as described above, with “mock” animals receiving 3 µl PBS on day 8 instead of influenza. Bacterial loads in nasal lavage fluid are shown. Each dot represents one mouse. Open circles designate index mice, and filled represent contact mice. Statistical significance was assessed using the Mann-Whitney test. Asterisk indicates <i>p</i><0.05.</p

TLR2 stimulation limits pneumococcal transmission.

<p><b>A.</b><i>tlr2^−/−</i> mice were infected as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004339#ppat-1004339-g001" target="_blank">Figure 1A</a>, and bacterial loads in nasal lavage fluid of contact mice are shown (filled triangles). Data from wildtype contacts (replicated from <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004339#ppat-1004339-g001" target="_blank">figure 1B</a>) are also shown for comparison (filled circles). <b>B.</b> Wildtype mice were infected as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004339#ppat-1004339-g001" target="_blank">Figure 1A</a>, and all pups were given Pam3Cys or PBS (vehicle control) intranasally on days 8, 10, and 12. Mice were sacrificed on day 14, and bacterial loads in lavage fluid of contact mice are shown. <b>C.</b> Age-matched wildtype and <i>tlr2^−/−</i> pups were split into index and contact groups and cross-fostered such that either the index were wildtype and the contacts were <i>tlr2^−/−</i> or that the index were <i>tlr2^−/−</i> and the contacts were wildtype. The infection scheme described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004339#ppat-1004339-g001" target="_blank">Figure 1A</a> was then followed, and bacterial loads in nasal lavage fluid are shown, with open symbols denoting index mice and filled symbols representing contact mice. ** indicates <i>p</i><0.01 by Mann-Whitney test.</p