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. 2009 Jul;1(4):211-22.
doi: 10.1002/emmm.200900025.

Streptococcus pneumoniae evades human dendritic cell surveillance by pneumolysin expression

Marie Littmann  1 , Barbara Albiger, Anne Frentzen, Staffan Normark, Birgitta Henriques-Normark, Laura Plant
Affiliations

Streptococcus pneumoniae evades human dendritic cell surveillance by pneumolysin expression

Marie Littmann et al. EMBO Mol Med. 2009 Jul.

Abstract (V体育平台登录)

Dendritic cells (DCs) protect the respiratory epithelium via induction of innate immune responses and priming of naïve T cells during the initiation of adaptive immunity. Streptococcus pneumoniae, a commonly carried asymptomatic member of the human nasopharyngeal microflora, can cause invasive and inflammatory diseases and the cholesterol-dependent cytotoxin pneumolysin is a major pneumococcal virulence factor implicated in compounding tissue damage and mediating inflammatory responses. While most studies examining the impact of pneumolysin have been based on murine models, we have focused this study on human DC responses. We show that expression of haemolytic pneumolysin inhibits human DC maturation, induction of proinflammatory cytokines and activation of the inflammasome. Furthermore, intracellular production of pneumolysin induces caspase-dependent apoptosis in infected DCs. Similarly, clinical isolates with non-haemolytic pneumolysin were more proinflammatory and caused less apoptosis compared to clonally related strains with active pneumolysin VSports手机版. This study describes a novel role of pneumolysin in the evasion of human DC surveillance that could have a profound clinical impact upon inflammatory disease progression and highlights the need to study human responses to human-specific pathogens. .

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Figures

Figure 1
Figure 1. Pneumococci are taken up and processed by DCs
A. DCs were infected with the encapsulated pneumococcal strain T4 (MOI 25, unless otherwise stated) and uptake was monitored by measuring intracellular bacteria by viable count after a 2 h infection period. An increased infection dose and opsonisation using pneumococcal antiserum to serotype 4 (anti-PN) increased the number of intracellular bacteria. The uptake of T4 was also compared to uptake of the unencapsulated mutant T4R (MOI 25). Significant increases compared to uptake of T4 (MOI 25, non-opsonised) are indicated by an asterisk (*). Data represent mean + SE (n = 3). B. Expression of pneumolysin aided the uptake of pneumococci by DCs. A significant decrease compared to the uptake of T4R is indicated by a double asterisk (**) and data represent mean + SE (n = 10). C. Quantification of the intracellular location of T4R and T4RΔply revealed that pneumococci reside in two types of vacuoles following uptake. Data represents the mean + standard deviation from two independent experiments. D. At 4 h post-infection T4R was detected in tight vacuoles in close contact with the cytosol, as indicated by asterisks. E. In contrast to T4R, after 4 h of infection T4RΔply resided both in spacious (arrows) and tight vacuoles (asterisks). F, G. After 8 h of infection, both strains of pneumococci were detected primarily in tight vacuoles (asterisks) but spacious vacuoles (arrows) were still observed in T4RΔply infected cells.
Figure 2
Figure 2. Uptake and intracellular pneumolysin production by pneumococci increase the apoptosis of DCs. DCs were infected for 24 h with T4 and mutants in capsule (T4R) and pneumolysin expression (T4Δply and T4RΔply)

  1. Statistical analysis of triplicate experiments revealed that T4R induced more apoptosis of human DCs than T4 as monitored by Annexin V-FITC positive cells (early apoptotic), Annexin V and PI positive cells (late-stage apoptotic) and PI positive cells (necrotic) V体育平台登录. Also, pneumolysin expression increased apoptosis irrespective of capsular expression. Mean (SE) is shown numerically and data is a compilation of three independent experiments. Significant changes compared to the parent strains (T4 and T4R) are indicated by p values. At least 5000 cells per treatment were counted by flow cytometry.

  2. Inhibition of uptake of T4R with cytochalasin D and wortmannin (CW) decreased cell death VSports注册入口. Data represents mean + SE from three independent experiments.

(V体育安卓版) Figure 3
Figure 3. Pneumolysin expression by live pneumococci inhibits the production of the proinflammatory cytokines IL-8 and IL-12 p70

  1. The effect of pneumolysin expression on the production of IL-12 p70 was examined following infection with live unencapsulated pneumococci at various MOIs. The production of IL-12 p70 was compared to cell viability by analysing the percentage of cells that stained negative for Annexin V and PI with flow cytometry. Data show mean ± SE (n = 3). Significant increases compared to T4R are indicated by an asterisk (*) for cytokines and a double asterisk (**) for cell viability.

  2. IL-8 production and cell viability was also examined following infection with live unencapsulated pneumococci at various MOIs. Data show mean ± SE (n = 3). Significant increases compared to T4R are indicated by an asterisk (*) for cytokines and a double asterisk (**) for cell viability.

  3. No effect of pneumolysin following infection with UV-killed pneumococci on the production of IL-12 p70 was observed. Data show mean ± SE (n = 3).

  4. Pneumolysin did not affect IL-8 or cell viability following infection with UV-killed pneumococci. Data represent mean ± SE (n = 3).

Figure 4 "VSports在线直播"
Figure 4. Pneumolysin expression activates multiple caspases in DCs

  1. Pneumolysin-proficient pneumococci significantly activated multiple caspases at 12 and 24 h post-infection. Data represents mean ± SE (n = 3). Significant increases compared to uninfected cells are indicated by an asterisk (*).

  2. Activation of caspase-1 at 24 h post-infection could also be attributed to pneumolysin expression. Data show mean + SE (n = 3) with significant differences indicated by an asterisk (*). 5000 events per treatment were counted by flow cytometry.

  3. The activation of caspase 3,7 at 24 h post-infection could be attributed to pneumolysin expression. Data represent mean + SE (n = 3) and significant differences are indicated by an asterisk (*). 5000 events per treatment were counted by flow cytometry.

Figure 5
Figure 5. Pneumolysin-deficient pneumococci induce the inflammasome-associated cytokine IL-1β

  1. Pneumolysin-deficient pneumococci activated caspase-1 at 6 h post-infection, albeit at non-significant levels. Data represent the mean + SE (n = 3). At least 5000 events per treatment were counted.

  2. In the absence of pneumolysin expression an increase in pro-IL-1β could be detected by Western blotting. Data is representative of three independent human DC preparations.

  3. Pneumolysin-deficient pneumococci enhanced secreted levels of mature IL-1β as early as 6 h post-infection and addition of the caspase-1 inhibitor Z-YVAD-FMK inhibited this secretion. Data represent the mean + SE (n = 3). A single asterisk (*) shows significant increases compared to T4R and a double asterisk (**) indicates significant decreases compared to values obtained without the inhibitor.

Figure 6
Figure 6. Pneumolysin expression diminishes DC activation
The effect of pneumolysin production on the expression of CD80 and CD86 by viable DCs was examined following infection with unencapsulated pneumococci. Data show the mean (% positive cells) + SE (n = 7). 10000 events per treatment were counted. Significant differences are indicated by an asterisk (*).
Figure 7
Figure 7. DC apoptosis, uptake and inhibition of IL-12 p70 induction following infection with invasive isolates of serotype 1 correlate with haemolytic pneumolysin expression

  1. DCs were infected for 24 h with invasive serotype 1 pneumococcal clinical isolates and stained with Annexin V-FITC and PI. Flow cytometry revealed that the isolates of ST217 (BHN 166), ST228 (BHN 32) and ST227 (BHN 418) which express haemolytic pneumolysin induced apoptosis, whereas isolates of ST306 (BHN 31, BHN 339 and BHN 349) which express non-haemolytic pneumolysin did not induce apoptosis. At least 5000 cells per treatment were counted by flow cytometry and data shows mean (SE) numerically (n = 3).

  2. Strains expressing non-haemolytic pneumolysin showed inhibited opsonised uptake. Data show mean + SE (n = 3).

  3. Strains expressing non-haemolytic pneumolysin increased IL-12 p70 induction. Data represent mean + SE (n = 3) and significant induction of IL-12 p70 is indicated by an asterisk (*).

Figure 8
Figure 8. Murine BMDCs respond differently to pneumococci than human DCs

  1. Human DCs showed enhanced uptake of pneumococci compared to murine BMDCs (BALB/c and C57BL/6). Data represent mean + SE. Significant decreases compared to human DCs are indicated by a single asterisk (*) and significant decreases compared to T4R are indicated by a double asterisk (**). Data comes from several independent experiments (human n = 10, BALB/c n = 4 and C57BL/6 n = 6).

  2. Murine BMDCs underwent similar apoptosis to human DCs following exposure to pneumolysin-proficient pneumococci, as detected by Annexin V-FITC and PI staining. Data represent mean + SE (n = 3). Significant decreases compared to T4R infected cells are shown with a double asterisk (**). At least 5000 cells per treatment were counted by flow cytometry.

  3. Human DCs showed increased production of the proinflammatory cytokine IL-12 p70 compared to BMDCs following pneumococcal infection. Data show mean + SE (n = 4). Significant increases compared to T4R infection are indicated by a single asterisk (*).

  4. Human DCs showed altered production of the proinflammatory cytokine IL-1β compared to BMDCs following pneumococcal infection. Data represent mean + SE (n = 4). Significant increases compared to T4R infection are indicated by a single asterisk (*) and significant decreases (p < 0.05) are indicated by a double asterisk (**).

Figure 9
Figure 9. Potential mechanisms involved in the human DC response to infection with pneumolysin-proficient and pneumolysin-deficient pneumococci
Infection with pneumolysin-proficient pneumococcal strains results in evasion of inflammatory responses (1–3). Following uptake, the pore-forming toxin pneumolysin induces rapid processing of bacteria into tight vacuolar compartments (1). Host sensing of bacterial components leads to an inhibition of cellular activation and cytokine secretion (2) and induction of caspase-dependent apoptosis of the DC (3). Infection of DCs with pneumolysin-deficient strains stimulates strong proinflammatory responses (I–IV). During early infection, bacteria reside in spacious vacuoles (I) and prolonged stimulation of vacuolar receptors leads to an enhanced immune response shown as up-regulation of CD80 and CD86 as well as synthesis of the cytokines IL-8 and IL-12 p70 (II). Signalling via an unknown vacuolar pattern recognition receptor (PRR) contributes to the up-regulation of pro-IL-1β (III). Inflammasome activation is triggered in the cell and early induction of active caspase-1 leads to processing of pro-IL-1β and pro-IL-18 and subsequent release of the mature proinflammatory cytokines IL-1β and IL-18 (IV).
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