Tolerability of novel nasal sampling methods.

<p>The percentage of volunteers rating (A) nasal curettage and (B) nasosorption on discomfort, pain and lacrimation.</p

Nasal curettage yields reproducible and consistent results over time.

<p>(A) The percentage of granulocytes (closed circles) and T cells (open circles) in 218 nasal cell samples collected over a five month period (n = 117 volunteers, sampled up to five times). Individual samples and loess curves are depicted for both populations. (B, C) The correlation for individuals in four repeated measurements over a 33-day period for (B) granulocytes and (C) T cells.</p

Comparison of samples collected by nasal wash and nasal curette.

<p>(A) Epithelial (open circles) and immune (closed circles) and cell yields were compared between nasal wash pellets and nasal curette samples. Individuals samples and median and interquartile range are shown. (B) Median proportions of granulocytes, T cells, monocytes, lineage^- HLA-DR⁺ and uncharacterized cells among immune cells in nasal curette (n = 139 individuals) and nasal wash (n = 8) samples. *p < 0.05, ***p < 0.001, ****p < 0.0001 Mann-Whitney test.</p

Comparison of samples from nasal mucosa and blood.

<p>(A) Median proportions of granulocytes, T cells, monocytes, lineage^- HLA-DR⁺ and uncharacterized cells among immune cells in blood (n = 10) and nasal curette (n = 139). **** p < 0.0001 Mann-Whitney test. (B) The percentage of HLA-DR⁺ T cells in blood and nasal curette samples and mean fluorescent intensity (MFI) of HLA-DR and CD66b on granulocytes was measured for blood, nasal curette and nasal wash (n = 8) samples. Median and interquartile range are shown. *p < 0.05, ***p < 0.001 Kruskal-Wallis, followed by Dunn’s Multiple Comparison Test. (C) Multi-dimensional scaling analysis shows the clustering of samples from blood (grey circles), nasal curette (open squares, 11 randomly selected) and nasal wash (black triangles). The epithelial cell yield, activation state of granulocytes and composition of the immune cells were taken into account. Kruskal stress = 5.8% and Analysis of Similarity ANOSIM p-value = 0.001.</p

Nasal Pneumococcal Density Is Associated with Microaspiration and Heightened Human Alveolar Macrophage Responsiveness to Bacterial Pathogens

Rationale: Pneumococcal pneumonia remains a global health problem. Colonization of the nasopharynx with Streptococcus pneumoniae (Spn), although a prerequisite of infection, is the main source of exposure and immunological boosting in children and adults. However, our knowledge of how nasal colonization impacts on the lung cells, especially on the predominant alveolar macrophage (AM) population, is limited. Objectives: Using a controlled human infection model to achieve nasal colonization with 6B serotype, we investigated the effect of Spn colonization on lung cells. Methods: We collected BAL from healthy pneumococcalchallenged participants aged 18-49 years. Confocal microscopy and molecular and classical microbiology were used to investigate microaspiration and pneumococcal presence in the lower airways. AM opsonophagocytic capacity was assessed by functional assays in vitro, whereas flow cytometry and transcriptomic analysis were used to assess further changes on the lung cellular populations. Measurements and Main Results: AMs from Spn-colonized individuals exhibited increased opsonophagocytosis to pneumococcus (11.4% median increase) for approximately 3 months after experimental pneumococcal colonization. AMs also had increased responses against other bacterial pathogens. Pneumococcal DNA detected in the BAL samples of Spncolonized individuals were positively correlated with nasal pneumococcal density (r = 0.71; P = 0.029). Similarly, AMheightened opsonophagocytic capacity was correlated with nasopharyngeal pneumococcal density (r = 0.61, P = 0.025). Conclusions: Our findings demonstrate that nasal colonization with pneumococcus and microaspiration prime AMs, leading to brisker responsiveness to both pneumococcus and unrelated bacterial pathogens. The relative abundance of AMs in the alveolar spaces, alongside their potential for nonspecific protection, render them an attractive target for novel vaccines