Splenic macrophages as the source of bacteraemia during pneumococcal pneumonia

Abstract

Background: Severe community-acquired pneumococcal pneumonia is commonly associated with bacteraemia. Although it is assumed that the bacteraemia solely derives from pneumococci entering the blood from the lungs it is unknown if other organs are important in the pathogenesis of bacteraemia. Using three models, we tested the relevance of the spleen in pneumonia-associated bacteraemia.

Methods: We used human spleens perfused ex vivo to explore permissiveness to bacterial replication, a non-human primate model to check for splenic involvement during pneumonia and a mouse pneumonia-bacteraemia model to demonstrate that splenic involvement correlates with invasive disease.

Findings: Here we present evidence that the spleen is the reservoir of bacteraemia during pneumonia. We found that in the human spleen infected with pneumococci, clusters with increasing number of bacteria were detectable within macrophages. These clusters also were detected in non-human primates. When intranasally infected mice were treated with a non-therapeutic dose of azithromycin, which had no effect on pneumonia but concentrated inside splenic macrophages, bacteria were absent from the spleen and blood and importantly mice had no signs of disease.

Interpretation: We conclude that the bacterial load in the spleen, and not lung, correlates with the occurrence of bacteraemia. This supports the hypothesis that the spleen, and not the lungs, is the major source of bacteria during systemic infection associated with pneumococcal pneumonia; a finding that provides a mechanistic basis for using combination therapies including macrolides in the treatment of severe community-acquired pneumococcal pneumonia.

Funding: Oxford University, Wolfson Foundation, MRC, NIH, NIHR, and MRC and BBSRC studentships supported the work.

Keywords: Bacteraemia; human organ; pathogenesis; pneumonia; sepsis; therapy.

Conflict of interest statement

Declaration of Competing Interest MRO reports grants from MRC MR/M003078/1, grants from BBSRC BB/S507052/1 , grants from MRC IMPACT Doctoral Training program, during the conduct of the study; grants from GSK co-funding of CASE studentship BBSRC BB/S507052/1, outside the submitted work; and consultancy for Pinsent Masons LLP 2020-21 on work unrelated to this manuscript. MRO had a BactiVac Network grant BVNCP3-05 2019-20 with GSK on topics unrelated to this manuscript. ZJ reports grants from BBSRC CASE GSK studentship BB/S507052/1, outside the submitted work. MP reports grants and personal fees from Gilead Sciences, personal fees from QIAGEN, grants from Sanofi, outside the submitted work. WSL has received core support from the NIHR Nottingham Biomedical Research Centre, and his institution has received unrestricted investigator-initiated research funding from Pfizer for an unrelated multicentre cohort study in which WSL is the Chief Investigator. The other authors have nothing to disclose.

Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.

Figures

Figure 1

Human spleen ex vivo perfusion and infection. Eight human spleens were perfused ex vivo with an artificial perfusate and infected with between 2 × 107 and 1.5 × 108 pneumococci. a) Bacterial counts in homogenised spleen biopsies were determined over the time of perfusion (three biopsies from each spleen at each time point). The eight infected perfusion experiments are colour coded HSP1 (red), HSP2 (blue), HSP5 (black), HSP6 (violet), HSP7 (green), HSP8 (pink), HSP9 (orange) and HSP12 (brown). b) The number of bacterial cells in each focus of infection was determined for at least 20 optical fields from at least 3 independent spleen sections in all perfused spleens for early (E; 0.5h) and late (L; 4 or 5h) time points. The breakdown into single spleen details is shown in Fig S1d. Statistical significance was determined by Fischer Exact test (***; p<0.001 * p=0.0324). c) Fluorescent scanning microscopy image of uninfected spleen HSP3 stained for cellular markers (DAPI nuclei, blue; CD206+ sinusoid cells of the red pulp, magenta; CD169+ macrophages, red). Some of the sliced periarteriolar sheaths composed of CD169+ macrophages at the border of the follicle (area devoid of CD206+ sinusoids) are indicated by arrows. The scale bar represents 300µm. d) Zoom of the dashed area of panel c. The scale bar represents 100µm. e-f) Independent representative clusters of pneumococci observed inside different CD169 positive splenic macrophages (red), imaged by confocal microscopy with 3D reconstruction of a Z-stack of microscopy images showing intracellular D39 pneumococci (green) with nuclei stained by DAPI (blue). The scale bars shown represent 5µm (e) and 10µm (f) respectively. All original image files, including a video of both 3-D reconstructions, are available at the Leicester data repository Figshare with the doi: 10.25392/leicester.data.12957947.v1.

Figure 2

Pneumococci in spleens of baboons during pneumonia. Spleen samples were obtained from stored material of a baboon pneumonia model with S. pneumoniae TIGR4 . a) Numbers of bacteria per splenic focus of infection in untreated (red) and ampicillin-treated (blue) baboons (n = 4 and 3 respectively). Bacterial counts were obtained from four random confocal microscopy fields. The number of macrophages with 2 or more bacteria was significantly higher in baboons B1-4 compared to those treated with ampicillin (B5-7), p<0.05. b) A representative image of CD163+ macrophages distribution in the red pulp of a baboon spleen (staining in all panels: bacteria, green; CD169+ macrophages, red; CD163+ red pulp macrophages, magenta; nuclei, blue). The scale bar represents 100µm. c) Perifollicular distribution of CD169+ macrophages. The scale bar represents 100µm. d) Clusters of pneumococcal foci in a non-treated baboon associated with CD163+ red pulp macrophages (magenta). The scale bar represents 10µm. e) Clusters of pneumococci were predominantly observed next to CD169+ peri-arteriolar sheath macrophages in the perifollicular area (CD169, red). The scale bar represents 20µm. f-g) Confocal and 3D reconstruction of the same Z-stack microscopy image showing that pneumococci are associated to CD169+ cells (CD169, red) (the resolution is lower than in Figure 1 due to the necessity for antigen retrieval in deparaffinised samples). The scale bars represent 20µm. h) Distribution of bacteria and their association with tissue macrophages in untreated, bacteraemic (red) and ampicillin-treated baboons (blue) determined using high throughput scanning fluorescence microscopy combined with semi-automated image analysis. Five areas were analysed for all seven baboon spleens. Statistical significance was determined by ANOVA (***, p<0.001; ns, not significant, p>0.05).

Figure 3

Pneumococci after intranasal challenge of mice and treatment with a low dose of azithromycin. a) Animals (n = 5) were intranasally challenged with 1 × 106 CFU of D39 pneumococci and after 30 min treated intraperitoneally with either a sub-inhibitory 1.128 μg/g BW azithromycin (open circle) or saline (closed circle). The antibiotic dose was chosen to be approximately 10x below the MIC in the serum. Mice were sampled at 6, 12, 24 or 48 hours after infection and bacterial counts were determined for lungs (blue), spleen (green) and blood (red). The limit of detection is shown as dotted line for the spleen and lung (green) and blood (red) samples. b) Signs of disease in mice were assessed and scored as 0 Normal, 1 Hunched, 2 Pilo-erection, 3 Loss of activity, 4 Lethargic, which was the severity limit of the experiment (filled circles: saline, open circles: azithromycin). c) Concentrations of azithromycin were determined by a bioassay of five homogenised spleens (green) and serum (red) of azithromycin-treated mice (values in Table S3). d) Effect of azithromycin treatment (same dose as panel a) on numbers of bacteria in the blood (red) and the spleen (green) after a separate intravenous challenge control experiment (n = 5; open circle azithromycin; closed circle saline). Statistical significance was determined by ANOVA (**, p<0.01; ***, p<0.001; ****, p<0.0001; ns not significant, p>0.05). e) 3D reconstruction of confocal image of CD169+ macrophages in the spleen at 24 h of a saline-treated mouse (CD169, red; bacteria, green; nuclei, blue). The scale bar represents 15µm.

Figure 4

Bacterial distribution and histology of the lungs after intranasal challenge in mice treated with a subinhibitory dose of azithromycin. Two groups of animals (n = 5) were intranasally infected with 1 × 106 CFU and, after 30 min, either treated intraperitoneally with low-dose azithromycin or saline. The animals were culled at 48 h and lungs were collected to analyse pneumococcal distribution and histology of the lungs. Haematoxylin and eosin staining, analysed blinded, showed no differences between control (a) and (b) azithromycin-treated mice, both groups showed the beginning of pneumonia with signs of inflammation in the subpleural area, the edge of the lungs and mesothelial cells. Increase in cellularity is indicated by an arrow. The scale bars represent 100µm. c) Whole lung tissue sections visualised by scanning microscopy. For each sample, 5 random areas were used to analyse bacterial distribution (squares). The scale bar represents 2mm. d) Distribution of bacteria in alveolar macrophages (AM, CD11c+ CD169+) interstitial macrophages (IM CD11c- CD169+) and in unstained tissue (other tissue) in azithromycin-treated (open bar) or saline-treated mice (filled bar) (five independent fields analysed for each of the 5 treatment and 5 control mice). Statistical significance was determined by ANOVA (*; p<0.05, ns; not significant). e) Representative phase contrast image of bacterial (in yellow/green) distribution within AM (magenta) and IM (red) in the lung using a 60x confocal objective. Arrows point to some of examples of AM containing bacteria and bacterial debris. The scale bar represents 20µm.