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

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 <i>iroA</i> locus protects against growth inhibition by lipocalin 2.

<p>Overnight growth in Lcn2-deficient mouse serum with or without 1.6 µM recombinant Lcn2 was determined for the <i>K. pneumoniae</i> mutants indicated (A). For complementation studies, growth in serum was determined for the <i>iroA</i> and <i>iroA ybtS</i> mutant transformed with the vector control (pACYC184) or pIroA (pACYC184::<i>iroBCDN</i>) (B). Mean±SEM for at least three independent experiments is shown as log₁₀ CFU/ml. Siderophores encoded by each mutant are indicated by a plus (+). *** p<0.001 comparing each condition to WT plus rLcn2, and # p<0.001 comparing pIroA to vector control, as determined by one-way ANOVA with Tukey's multiple comparison test. To determine if wild-type <i>K. pneumoniae</i> produces Lcn2-resistant and sensitive Ent, growth of the <i>entB</i> mutant in serum with or without 5 µM recombinant Lcn2 was compared between vehicle control, 16 nM of purified Ent or Gly-Ent (Salmochelin S4), or wild-type culture supernatant (1∶640 dilution) (C). Mean ± standard deviation for two independent experiments is shown as log₁₀ CFU/ml.</p

Mucosal lipocalin 2 is required to inhibit colonization by enterobactin-dependent <i>K. pneumoniae</i>.

<p>Colonization density of <i>Lcn2^+/+</i> (shaded symbols) and <i>Lcn2^−/−</i> mice (open symbols) on day 3 after intranasal inoculation with 2×10⁶ cfu of the <i>K. pneumoniae</i> mutants indicated (A). Box and whiskers graph shows the median and interquartile ranges for ≥10 mice per group; the dashed line is the lower limit of detection (20 cfu/ml). Siderophores encoded by each mutant are indicated by a plus (+). * p<0.05, ** p<0.01, ^ns p>0.05 as determined by Kruskall-Wallis Test with Dunn's post test. To remove the contribution of neutrophil-produced Lcn2, colonization density on day 1 was determined in <i>Lcn2^+/+</i> (n = 6) and <i>Lcn2^−/−</i> mice (n = 8) treated with RB6-8C5 rat mAb to murine Ly6G one day prior to inoculation with 2×10⁶ cfu of <i>iroA ybtS</i> mutant <i>K. pneumoniae</i> (B). * p<0.05 as determined by Mann-Whitney Test.</p

Neutrophils inhibit <i>K. pneumoniae</i> colonization and prevent bacteremia.

<p>Mice were treated with RB6-8C5 rat mAb to murine Ly6G or control rat IgG one day prior to inoculation with 2×10⁶ cfu of wild-type <i>K. pneumoniae</i> KPPR1 were sacrificed at day 1 post-inoculation. Nasopharyngeal lavage cfu/ml (A) and spleen cfu/gm (B) are shown as a scatter plot with the bar at the median; dashed line represents lower limit of detection. ** p<0.01 by Mann-Whitney test; * p<0.05 by Fisher's Exact Test for presence of detectable bacteria in the spleen.</p

<i>K. pneumoniae</i> colonization elicits an acute inflammatory infiltrate.

<p>Mice colonized with wild-type <i>K. pneumoniae</i> KPPR1 were sacrificed at day 1 post-inoculation. Inflammatory infiltrates are seen adjacent to the turbinates of the nasopharynx (A, 20×, arrows) containing neutrophils (B, 600×, arrowheads). To enumerate the neutrophil influx, mice were sacrificed at day 3 post-inoculation for nasopharyngeal lavage, and 100 µL of lavage fluid was analyzed by flow cytometry. Neutrophils (CD45+, Ly6G+ and CD11b+) from a representative KPPR1 (C, D) or PBS mock-colonized (E, F) mouse lavage sample are shown. Compiled neutrophil counts are shown as a scatter plot; the bars represent median numbers of CD45+, Ly6G+, CD11b+ events/ml for each animal (G). p<0.01 by Mann-Whitney test.</p

<i>K. pneumoniae</i> colonizes the nasopharynx of C57BL/6 mice.

<p>C57BL/6 mice were inoculated intranasally with 2×10⁶ cfu of an overnight LB culture of KPPR1 in 10 µL PBS. At each time point, 5 mice were sacrificed, nasopharyngeal lavage was performed, and lavage fluid was plated for quantitative culture. Colony forming unit (cfu) counts are shown as a scatter plot where the bar represents the median; the dashed line is the lower limit of detection (20 cfu/ml).</p

Enterobactin-dependent <i>K. pneumoniae</i> is defective in colonization.

<p>Colonization density at day 3 after intranasal inoculation with 2×10⁶ cfu of the <i>K. pneumoniae</i> mutants indicated was determined in C57BL/6 mice (n≥5 mice per group). Box and whiskers graph shows the median and interquartile ranges; the dashed line is the lower limit of detection (20 cfu/ml). Siderophores encoded by each mutant are indicated by a plus (+). *** p<0.001 as determined by Kruskall-Wallis Test with Dunn's post test.</p