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. 2018 Aug 22;86(9):e00186-18.
doi: 10.1128/IAI.00186-18. Print 2018 Sep.

Platelets Enhance Dendritic Cell Responses against Staphylococcus aureus through CD40-CD40L

Sharmeen Nishat  1 , Leah M Wuescher  1 , Randall G Worth  2
Affiliations

Platelets Enhance Dendritic Cell Responses against Staphylococcus aureus through CD40-CD40L

Sharmeen Nishat et al. Infect Immun. .

Abstract

Staphylococcus aureus is a major human pathogen that can cause mild to severe life-threatening infections in many tissues and organs. Platelets are known to participate in protection against S. aureus by direct killing and by enhancing the activities of neutrophils and macrophages in clearing S. aureus infection. Platelets have also been shown to induce monocyte differentiation into dendritic cells and to enhance activation of dendritic cells. Therefore, in the present study, we explored the role of platelets in enhancing bone marrow-derived dendritic cell (BMDC) function against S. aureus We observed a significant increase in dendritic cell phagocytosis and intracellular killing of a methicillin-resistant Staphylococcus aureus (MRSA) strain (USA300) by thrombin-activated platelets or their releasates. Enhancement of bacterial uptake and killing by DCs is mediated by platelet-derived CD40L. Coculture of USA300 and BMDCs in the presence of thrombin-activated platelet releasates invokes upregulation of the maturation marker CD80 on DCs and enhanced production of the proinflammatory cytokines tumor necrosis factor alpha (TNF-α), interleukin 12 (IL-12), and IL-6. Overall, these observations support our hypothesis that platelets play a critical role in the host defense against S. aureus infection. Platelets stimulate DCs, leading to direct killing of S. aureus and enhanced DC maturation, potentially leading to adaptive immune responses against S. aureus.

Keywords: CD40L; S. aureus; dendritic cells; phagocytosis; platelets.

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Figures

FIG 1
FIG 1
Effect of thrombin-activated platelets on DC phagocytosis and killing of S. aureus. BMDCs were incubated with MRSA (at an MOI of 10) in sterile DPBS−/− (black bars) in the presence or absence of unstimulated (white bars) or thrombin-activated (gray bars) platelets for 1 h (t = 1). The DCs were washed with DPBS−/−, lysed with Triton, and plated on TSA plates to obtain internalized CFU. (A) MRSA CFU obtained from DC lysates (t = 1). (B to D) DCs from panel A (bars of corresponding shades) were incubated for a further 3 h (t = 4) in the presence or absence of thrombin-activated platelets. Intracellular bacteria were obtained from lysed DCs, and CFU were enumerated. All the values are presented as means and standard deviations (SD) (each experiment was performed in triplicate samples and repeated on at least 3 different days).
FIG 2
FIG 2
Effect of thrombin-activated platelet releasates on DC phagocytosis and killing of S. aureus. (A) Representative confocal images of DCs (t = 1 h). BMDCs were incubated with FITC-labeled MRSA in RPMI 1640 medium in the presence or absence of thrombin-activated platelet releasates for 1 h (t = 1), washed with DPBS−/−, and stained with TRITC-conjugated anti-IgG secondary Ab. (B) BMDCs were incubated with MRSA (at an MOI of 10) in sterile DPBS−/− in the presence or absence of thrombin-activated platelet releasates for 1 h (t = 1). The DCs were washed with DPBS−/−, lysed with Triton, serially diluted, and plated on TSA plates to obtain internalized CFU (t = 1). (C and D) DCs from panel B were further incubated for 3 h (t = 4) in the presence or absence of thrombin-activated platelets. Intracellular bacteria were obtained from lysed DCs, and CFU were measured. (E) For inhibition of phagocytosis, DCs were incubated with cytochalasin D at a 200 μM final concentration or an equal volume of DPBS−/− for 30 min at 37°C prior to phagocytosis assay. CFU were measured for bacteria obtained from lysed DCs. All measures are presented as means and SD (each experiment was performed in triplicate samples and repeated on at least 3 different days).
FIG 3
FIG 3
Effect of sCD40L on DC phagocytosis and killing of S. aureus. (A) BMDCs were incubated with MRSA (at an MOI of 10) in sterile DPBS−/− in the presence of sCD40L, RANTES, IL-1β, or DPBS−/− for 1 h (t = 1). The DCs were washed with DPBS−/−, lysed with Triton, and plated on TSA plates to obtain internalized CFU. (B) BMDCs were incubated with MRSA (at an MOI of 10) in sterile DPBS−/− in the presence or absence of thrombin-activated platelet releasates or sCD40L for 1 h (t = 1). The DCs were washed with DPBS−/−, lysed with Triton, and plated on TSA plates to obtain internalized CFU. (C) DCs (panel B, first bar from left) were further incubated for 3 h (t = 4) in the presence or absence of thrombin-activated platelet releasates or sCD40L. Intracellular bacteria were recovered from lysed DCs, and CFU were measured. (D) DCs were pretreated with anti-CD40 antibody or DPBS−/−, followed by incubation with MRSA (at an MOI of 10) in sterile DPBS−/− in the presence or absence of thrombin-activated platelet releasates for 1 h. Bacteria obtained from lysed DCs were plated, and CFU were enumerated. All measures are presented as means and SD (each experiment was performed in triplicate samples and repeated on at least 3 different days).
FIG 4
FIG 4
Effect of thrombin-activated platelet releasates on DC activation. (A) BMDCs were incubated with MRSA (at an MOI of 10) in sterile DPBS−/− in the presence or absence of thrombin-activated platelet releasates for 3 h. The cells were harvested, washed with DPBS−/−, and stained with PE-CD11c Ab and FITC-CD80 Ab for flow cytometry analysis. Mean fluorescence intensity (MFI) values were obtained by analyzing the data using FlowJo. Cells were gated on CD11c+ dendritic cells and analyzed for expression of CD80. (B to D) BMDCs were incubated with MRSA (at an MOI of 10) in sterile DPBS−/− in the presence or absence of thrombin-activated platelet releasates for 2 h. T cell media containing 1% pen-strep were added to the cells and incubated overnight. Cytokines in the culture supernatants were quantified by ELISA. All measures are presented as means and SD (each experiment was performed in triplicate samples and repeated on 3 different days).
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