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

Influenza infection induces bacterial shedding at a level high enough to infect infant mice.

<p><b>A–E.</b> Wildtype (A, C, and E) and <i>tlr2^−/−</i> mice (B and D) were infected with either <i>S. pneumoniae</i> and influenza (A, B and E) or <i>S. pneumoniae</i> alone (C and D) 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>. Each day (starting on day 8), the nose of each mouse was gently pressed onto an agar plate 10 times, exhaled bacteria were spread, and plates were incubated overnight. Each symbol represents the bacterial count from one animal on each day. In panel E, Pam3Cys or PBS alone (vehicle control) was administered on days 8, 10 and 12. Colony counts from days 10 and 11 are combined for comparison. <b>F.</b> Infant (7 day old) and adult (6 week old) mice were inoculated with ∼500 (dotted line) CFU of strain P1121 in PBS. One day post inoculation, the mice were sacrificed, nasal lavage was performed and lavage fluid was serially diluted and plated on selective media to enumerate bacteria.</p