Pneumococcal Extracellular Vesicles Modulate Host Immunity

Abstract

Extracellular vesicles (EVs) have recently garnered attention for their participation in host-microbe interactions in pneumococcal infections. However, the effect of EVs on the host immune system remain poorly understood. Our studies focus on EVs produced by Streptococcus pneumoniae (pEVs), and reveal that pEVs are internalized by macrophages, T cells, and epithelial cells. In vitro, pEVs induce NF-κB activation in a dosage-dependent manner and polarize human macrophages to an alternative (M2) phenotype. In addition, pEV pretreatment conditions macrophages to increase bacteria uptake and such macrophages may act as a reservoir for pneumococcal cells by increasing survival of the phagocytosed bacteria. When administered systemically in mice, pEVs induce cytokine release; when immobilized locally, they recruit lymphocytes and macrophages. Taken together, pEVs emerge as critical contributors to inflammatory responses and tissue damage in mammalian hosts. IMPORTANCE Over the last decade, pathogen-derived extracellular vesicles (EVs) have emerged as important players in several human diseases. Therefore, a thorough understanding of EV-mediated mechanisms could provide novel insights into vaccine/therapeutic development. A critical question in the field is: do pathogen-derived EVs help the pathogen evade the harsh environment in the host or do they help the host to mount a robust immune response against the pathogen? This study is a step towards answering this critical question for the Gram-positive pathogen, Streptococcus pneumoniae. Our study shows that while S. pneumoniae EVs (pEVs) induce inflammatory response both in vitro and in vivo, they may also condition the host macrophages to serve as a reservoir for the bacteria.

Keywords: EVs; Gram-positive bacteria; Streptococcus pneumoniae; alternative activation pathway; extracellular vesicles; host-pathogen interactions; immune response; macrophage signaling; pathogenesis.

FIG 1

Viable pneumococci produce EVs. (A) Representative transmission electron micrograph image of pEVs purified by SEC from strain R6. (B) Representative output from nanoparticle tracking analyses of pEVs, displaying size distribution and concentration. Bacterial cells used for purification were grown (i) in rich media or (ii) in rich media supplemented with 2% choline chloride to inhibit autolysis (n = 2).

FIG 2

pEVs are internalized by host cells. (A) Representative confocal images of A549 lung epithelial cells (left panels) and murine macrophages (J774A.1; right panels) exposed to pEVs for the indicated times. Red, DiD-stained pEVs; green, F-actin; blue, DAPI nuclear stain. (B) Representative flow cytometry measurements of the amounts of DiD detected in cells before the addition of pEVs (control) and at five time points after the addition of 20 μg/ml of pEVs (n = 2). The red line demonstrates the fluorescence intensity of cells without any pEVs. All cells exhibited pEV internalization by 24 h.

FIG 3

pEVs induce NK-κB signaling in macrophages. (A) Graph of NF-κB production in response to various concentrations of pEVs contrasted to negative (no pEVs; untreated) and positive (0.1 μg/ml LPS) controls. Measurements from a murine macrophage reporter cell line where secreted fetal alkaline phosphatase is under the control of NF-κB. The supernatant of the cell line was assayed for reporter activity 24 h postexposure to pEVs representing cumulative reporter release and overall NF-κB activation (n = 2). (B) Representative confocal images of NF-κB activation in primary human macrophages, 30 min after exposure to pEVs (20 μg/ml) or control groups (n = 2). Blue, DAPI nuclear stain; white, p65.

FIG 4

Systemic inoculation of pEVs in mice induces immune changes. (A and B) Cell markers were used to assess changes in PBMC and splenocyte populations. The cell types assessed were as follows: regulatory T cells (CD4⁺ and FoxP3⁺), helper T cells (CD3⁺, CD45⁺, and CD4⁺), cytotoxic T cells (CD3⁺, CD45⁺, and CD8⁺), all macrophages (CD45⁺, CD11b⁺, and F480⁺), M1 macrophages (CD45⁺, CD11b⁺, F480⁺, CD86⁺, and CD80⁺), M2 macrophages (CD45⁺, CD11b⁺, F480⁺, and CD206⁺), natural killer cells (CD45⁺ and NKp46⁺), and myeloid-derived suppressor cells (CD45⁺, CD11b⁺, and Gr1⁺). (A) Mice were inoculated intravenously with pEVs purified from wild-type bacteria (0.1 ml with 15 μg of pEVs) and analyzed for relative changes in PBMC populations preinoculation and 24 h postinoculation (n = 11 animals per cohort over two independent experiments). FC, fold change. (B) Postinoculation PBMCs identified as percent positive populations, comparing mice inoculated with pEVs to PBS control group. (C) Postinoculation splenocyte identified as percent positive populations, comparing pEV inoculations to PBS control group. (B and C) Box-and-whisker plots show median, range 25 to 75%, and min-max values and n = 11 for pEV and for control groups. Data were collected over two independent experiments. *, P ≤ 0.05; **, P ≤ 0.01 (versus control group). (D) Gross anatomy of excised spleens from the first experimental set of eight animals. (i) Red dashed ellipsoids highlight macroscopic legions, (ii) whereas a yellow arrow denotes a region with necrosis. (E) Representative histological images of spleens from mice treated with PBS (left panels) or pEVs (right panels). Within each group, the left image is stained with H&E, whereas the right upper panel represents a magnified region from the green dashed rectangle of the corresponding left panel. The yellow arrow identifies tingible body macrophages. The right lower panel is a representative image of immunostaining with CD68, a general macrophage marker.

FIG 5

Lipoproteins associated with pEVs contribute to macrophage signaling and alter PBMC landscape. (A) NF-κB production in murine macrophage reporter cell line (RAW-Blue) is reduced for Δlgt-pEVs compared to pEVs. Bars represent the means ± the standard errors of the mean (SEM) (n = 3). ****, P ≤ 0.0001 (versus control group); #, P ≤ 0.0001 (versus pEV group). (B) Cell markers were used to determine the nature and the relative quantification of PBMCs and splenocytes. The cell types assessed were defined as follows: regulatory T cells (CD4⁺ and FoxP3⁺), helper T cells (CD3⁺, CD45⁺, and CD4⁺), cytotoxic T cells (CD3⁺, CD45⁺, and CD8⁺), all macrophages (CD45⁺, CD11b⁺, and F480⁺), M1 macrophages (CD45⁺, CD11b⁺, F480⁺, CD86⁺, and CD80⁺), M2 macrophages (CD45⁺, CD11b⁺, F480⁺, and CD206⁺), natural killer cells (CD45⁺ and NKp46⁺), and myeloid-derived suppressor cells (CD45⁺, CD11b⁺, and Gr1⁺). Postinoculation PBMCs are identified as percent positive populations, comparing mice inoculated with pEVs from the wild type, pEVs from the Δlgt strain, and the PBS control group. Asterisks (*) represent statistical significance relative to pEV from wild-type cells, and number symbols (#) represent statistical significance relative to controls. Box-and-whisker plots show median, range 25 to 75%, and min-max values (n = 11 per cohort). (C) Violin plots (n = 11 per cohort) showing relative percentages of PBMC pre- and posttreatment with wild-type pEVs (gray) and Δlgt-pEVs (white). *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.

FIG 6

Systemic inoculation of pEVs triggers induction of immune markers in mice. (A) Heat map of 40 immune markers in mouse plasma. Mice were inoculated intravascularly with PBS control, pEVs (15 μg in 100 μl), or Δlgt-pEVs (15 μg in 100 μl). Experiments were performed in triplicate, each from a set of pooled plasma from three mice. (B) Venn diagram of data highlighting the number of cytokines where the concentration was changed between pre- and postinoculation (heat map value 1 to 5) in each group (independent of mice sex). (C) Heat map of 13 cytokines upregulated in plasma, displayed considering sex as a biological variable where male and female mice are plotted separately. Data from the females represent the average of two cohorts, while data from males represent one cohort. Mice were inoculated intravascularly with PBS, pEVs, or Δlgt-pEVs. The ELISA blots are presented in Fig. S3.

FIG 7

Local inoculation of pEVs in mice. Cohorts of 12 mice each, over two independent experiments, were injected with growth factor-reduced basement membrane extract hydrogel (0.5 ml) containing PBS control (0.1 ml), pEVs purified from wild-type bacteria (15 μg in 0.1 ml of pEVs), or Δlgt-pEVs purified from the Δlgt strain (15 μg in 0.1 ml of Δlgt-pEVs) and analyzed 7 days postinoculation. (A) Experimental design schematic, where the red circle highlights the hydrogel at the experimental endpoint. (B) Photograph of hydrogel plugs harvested from the first independent experiment. In one PBS-treated female, the hydrogel was no longer present at the site of injection, and this animal was removed from the analysis. (C) Representative images of hydrogel slices stained with H&E. Top panels show images at the interface between the skin and the plug; bottom panels show magnified zones from the regions denoted in the left panels. (D to F) Cell markers were used to assess levels of different cell types using flow cytometry. The cell types were defined as follows: regulatory T cells (CD4⁺ and FoxP3⁺), helper T cells (CD3⁺, CD45⁺, and CD4⁺), cytotoxic T cells (CD3⁺, CD45⁺, and CD8⁺), all macrophages (CD45⁺, CD11b+, and F480+), M1 macrophages (CD45+, CD11b+, F480+, CD86+, and CD80+), M2 macrophages (CD45+, CD11b+, F480+, and CD206+), natural killer cells (CD45+ and NKp46+), and myeloid-derived suppressor cells (CD45+, CD11b+, and Gr1+). (D) Relative quantification of infiltrated cells in pEV and Δlgt-pEV hydrogel implant. For each cohort, implants from 12 animal were pooled into three groups to get enough cells for flow cytometry analysis. Bars indicate means ± the SEM (n = 3). (E and F) Changes in the relative percentages of PBMCs and splenocytes pre- and postinjection of hydrogel containing PBS or pEVs or Δlgt-pEV. Box-and-whisker plots show median, range 25 to 75%, and min-max values (n = 12).

FIG 8

pEVs induce alternatively activated macrophages. Primary human macrophages were exposed to live bacteria (MOI = 5) or 20 μg/ml pEVs or 20 μg/ml Δlgt-pEV. To differentiate between the M1 macrophage phenotype and the M2 macrophage phenotype, cells were treated with 10 ng/ml LPS or 50 ng/ml IL-4, respectively, as controls. Graphs indicate the percent positive cells for the indicated cell markers that include classically activated “inflammatory” macrophages (IFN-γ and CD80 positive) or alternatively activated “immunoregulatory” macrophages (Arg-1 and IL-10 positive). (A) Response to wild-type bacteria and pEVs. (B) Response to Δlgt bacteria and Δlgt-pEVs. Bars represent means ± the SEM (n = 3). (C) Bar graph showing an assessment of bacteria phagocytosed by different subtypes of primary human macrophages. Bars indicate cells positive for phagocytosed bacteria after 1 h of infection with SYTO-9-labeled bacteria. Bars represent means ± the SEM (two independent experiments with n = 3). (D) Percentages of surviving bacterial cells (by viable plating) at different time points after internalization in macrophages. Points indicate means ± the SEM (n = 3). Black dotted line indicates the survival trend curve with slope representing the rate of bacterial death in macrophages.

FIG 9

Pneumolysin contributes to pEV-induced macrophage activation and phagocytosis. (A) NF-κB production in murine macrophage reporter cell line (RAW-Blue) is reduced for Δply-pEVs compared to pEVs. Bars represent the means ± the SEM of three independent experiments (n = 3; all conditions run in parallel). ****, P  ≤ 0.0001; ***, P  ≤ 0.001 (versus control group); #, P  ≤ 0.01 (versus pEV group). (B) Percentages of surviving bacterial cells (by viable plating) at different time points after internalization in macrophages. Points indicate means ± the SEM (n = 4). The dotted black line indicates the survival trend curve, with the slope representing the rate of bacterial death in macrophages. (C) Bar graph showing slopes of bacteria survival plot shown in panel B. The percentages of viable and internalized bacteria over time were fitted to a linear curve. The slope of the fitted trend line was used as an estimate for rate of bacterial death. Slopes of individual experiments are depicted as dots. Bars represent means ± the SEM (n = 4).