Platelets protect from septic shock by inhibiting macrophage-dependent inflammation via the cyclooxygenase 1 signalling pathway

Abstract

Although it has long been known that patients with sepsis often have thrombocytopenia and that septic patients with severe thrombocytopenia have a poor prognosis and higher mortality, the role of platelets in the pathogenesis of sepsis is poorly understood. Here we report a protective role of platelets in septic shock. We show that experimental thrombocytopenia induced by intraperitoneal injection of an anti-glycoprotein Ibα monoclonal antibody increases mortality and aggravates organ failure, whereas transfusion of platelets reduces mortality in lipopolysaccharide-induced endotoxemia and a bacterial infusion mouse sepsis model. Plasma concentrations of proinflammatory cytokines TNF-α and IL-6 are elevated by thrombocytopenia and decreased by platelet transfusion in septic mice. Furthermore, we identify that platelets protect from septic shock by inhibiting macrophage-dependent inflammation via the COX1/PGE₂/EP4-dependent pathway. Thus, these findings demonstrate a previously unappreciated role for platelets in septic shock and suggest that platelet transfusion may be effective in treating severely septic patients.

Figure 1. Depletion of platelets in mice enhances mortality and worsens organ failure induced by LPS

(a) C57BL/6 mice were injected with 4 µg/g of body weight of a rat anti-mouse GPIbα monoclonal antibody (n = 7) or control rat IgG (n = 8) by i.p. Platelets counts were measured with a HEMAVET HV950FS multispecies hematology analyzer before and 4 hours after injection of antibody. Values are means ± s.d. (b) Mice were then injected with LPS (10 mg/kg). Survival rate was observed for up to 3 days. P value was calculated by Wald test in a discrete time hazard model using version 9.2 of SAS software. (c–f), 8 hours after LPS treatment, plasma from mice receiving anti-GPIb antibody or IgG control were collected and ALT (c), AST (d), LDH (e), and CK (f) concentrations in plasma were measured. Values are means ± s.d. (n = 4). Differences between two groups were assessed using unpaired two-tailed Student’s t-test. (g,h) Mice were injected intraperitoneally with a rat anti-mouse GPIbα monoclonal antibody or rat IgG control. After 4 hours, the mice were injected intraperitoneally with LPS (10 mg/kg). Fifteen hours after LPS injection, the mice were sacrificed and lungs were collected (g). Sections of lungs were stained with H&E and images were captured. Scale bar, 0.5 mm. (h).

Figure 2. The effects of thrombocytopenia on plasma TNF-α and IL-6 concentrations

(a,b) Mice were injected intraperitoneally with a rat anti-mouse GPIbα monoclonal antibody or rat IgG control. After 4 hours, the mice were injected intraperitoneally with LPS (10 mg/kg). Blood was collected from these mice before orat 1, 2.5, 4, and 8 hours after LPS injection. TNF-α (a) and IL-6(b) concentrations in plasma were measured by an ELISA assay. Values are means ± s.d. (n = 6). Differences between two groups were assessed using unpaired two-tailed Student’s t-test. (c,d) Mice were injected retro-orbitally with clodronate (40 mg/kg) or control liposome. Blood was collected from the mice at 24 hours after injection of clodronate or liposome. Representative flow cytometry plots of monocyte subsets (c) and quantification of monocyte subsets (d) (n = 3) are shown. Error bars indicate s.d. Difference was assessed using unpaired two-tailed Student’s t-test. (e,f) Mice were injected retro-orbitally with clodronate (40 mg/kg) or control liposomes at 24 and 4 hours prior to LPS injection. The mice were also injected intraperitoneally with a rat anti-mouse GPIbα monoclonal antibody or rat IgG control at 4 hours prior to LPS injection. Blood was collected before or at 1, 2, 4, and 8 hours after LPS injection, and plasma TNF-α (e) and IL-6 (f) concentrations were measured. Values are means ± s.d. (n = 5). Differences between two groups were assessed using unpaired two-tailed Student’s t-test.

Figure 3. Platelets inhibit inflammation and protect against septic shock in a bacterial infusion sepsis model

(a,b) Mice were injected intraperitoneally with a rat anti-mouse GPIbα monoclonal antibody or rat IgG control. After 4 hours, the mice were injected intraperitoneally with 0.2 mL saline containing 2 × 10⁷ CFU of E. coli ATCC 25922 per mouse. Blood was collected from these mice before or at 1, 2, 4, and 8 hours after injection of bacteria. TNF-α (a) and IL-6 (b) concentrations in plasma were measured by ELISA assays. Values are means ± s.d. (n = 6). Differences between two groups were assessed using unpaired two-tailed Student’s t-test. (c) C57BL/6 mice were injected intraperitoneally with a rat anti-mouse GPIbα monoclonal antibody or control rat IgG (4 µg/g of body weight). After 4 hours, the mice were injected with 0.2 mL saline containing 2 × 10⁷ CFU of E. coli ATCC 25922. Survival rate was observed for up to 3 days. P value was calculated by Wald test in a discrete time hazard model using version 9.2 of SAS software.

Figure 4. The effects of platelet transfusion on plasma TNF-α and IL-6 concentrations and mortality in septic animals

(a–c) Mice were injected with LPS i.p. (10 mg/kg) followed by retro-orbital injection of 1 × 10⁹ platelets in 0.2 ml saline or saline. Blood were collected before or at 1, 2.5, 4, and 8 hours after LPS injection. Platelets counts were measured before or at 1 and 8 hours after LPS injection and expressed as relative to the counts of saline-treated mice before injection of LPS (a). Plasma TNF-α (b) and IL-6 (c) concentrations were measured. Values are means ± s.d. (n = 5). Differences between two groups were assessed using unpaired two-tailed Student’s t-test. (d) Mice were injected with LPS (10 mg/kg) immediately followed by retro-orbital injection of 1 × 10⁹ platelets in 0.2 ml saline or saline. Survival rate was observed for 3 days. P value was calculated by Wald test in a discrete time hazard model using version 9.2 of SAS software. (e) C57BL/6 mice were injected intraperitoneally with 0.2 mL saline containing 2.5 × 10⁷ CFU of E. coli ATCC 25922 immediately followed by retro-orbital injection of 1 × 10⁹ platelets in 0.2 ml saline or saline. Survival rate was observed for up to 3 days. P value was calculated by Wald test in a discrete time hazard model using version 9.2 of SAS software. (f) Mice were injected retro-orbitally with clodronate or control liposomes at 24 and 4 hours prior to LPS injection (5 mg/kg of body weight), immediately followed by retro-orbital injection of 1 × 10⁹ platelets in 0.2 ml saline or saline. Survival rate was observed for up to 3 days. p<0.001 between liposome group and clodronate group; p>0.2 between clodronate group injected with saline and clodronate group injected with platelets. P value was calculated by Wald test in a discrete time hazard model using version 9.2 of SAS software.

Figure 5. Platelets or platelet releasates inhibit TNF-α and IL-6 production induced by LPS in bone marrow-derived macrophages (BMDMs)

(a) LPS (1 ng/ml~10 µg/ml) was added with buffer or washed platelets (3 × 10⁸/ml) to BMDMs and incubated for 6 hours. TNF-α and IL-6 concentrations in the supernatant were measured. (b) Washed platelets or medium were incubated with ³H-LPS (250 ng/ml) at 37°C for 2 hours and centrifuged. DPMI in medium, platelet pellet or supernatant was measured. (c) LPS (100 ng/ml) was added to BMDMs in the presence or absence of platelets (3 × 10⁸/ml), and incubated for 6 hours. Washed platelets (3 × 10⁸/ml) were also incubated with LPS (100 ng/ml) for 30 min or thrombin (Enzyme Research Laboratories, South Bend, IN, 0.1 U/ml) in aggregometer at 37°C for 5 min and centrifuged. The resulting supernatants with LPS were added to BMDMs and incubated for 6 hours. LPS plus thrombin (Throm) were added to BMDMs and incubated for 6 hours as a control. TNF-α and IL-6 concentrations in the supernatant were measured. (d) Washed platelets from G_q knockout mice or wild-type littermates were incubated with thrombin in aggregometer for at 37°C 5 min and centrifuged. Supernatants plus LPS were added to BMDMs and incubated for 6 hours. (e) Platelets (3 × 10⁸/ml) were preincubated with thrombin in aggregometer at 37°C for 5 min and centrifuged. SAA (1 µg/ml) together with buffer or supernatant from thrombin-stimulated platelets were added to BMDMs and incubated for 6 hours. Values are means ± s.d. (n = 3 for all). Differences between two groups were assessed using unpaired two-tailed Student’s t-test.

Figure 6. Platelets inhibit TNF-α production by macrophages through the COX1/PGE₂/EP4 pathway

(a) Washed platelets were pre-incubated with aspirin (1 mM) or buffer for 5 min and then incubated with thrombin in aggregometer at 37°C for 5 min and centrifuged. LPS plus the supernatants of platelets were added to BMDMs and incubated for 6 hours. Thrombin or thrombin plus aspirin were added to the wells with LPS in the absence of platelet supernatant as controls. (b) Platelets from COX1 knockout mice (COX1^−/−) or wild-type littermates (COX1^+/+) were incubated with buffer (resting) or thrombin in aggregometer at 37°C for 5 min. LPS plus the supernatants of platelets were added to BMDMs and incubated for 6 hours. (c) Washed platelets from C57BL/6J mice were incubated with LPS for 30 min or thrombin in aggregometer at 37°C for 5 min. PGE₂ in the supernatant were measured by an ELISA assay (Cayman Chemical, Ann Arbor, MI). (d) BMDMs were pre-incubated with buffer, SC -51089 10 µM, AH 6809 10 µM, pertusis toxin (PTX) 0.5 µg/ml, or GW627368X (GW62) at 37°C for 15 min. BMDMs were then added with LPS or LPS plus supernatant from thrombin-activated platelets and incubated at 37°C for 6 hours. Values are means ± s.d. (n = 3 for all). Differences between two groups were assessed using unpaired two-tailed Student’s t-test.

Figure 7. The effects of COX1 knockout and aspirin on platelet-dependent inhibition of inflammation and protection against sepsis

(a,b) C57BL/6 mice received intraperitoneal injection of LPS (10 mg/kg) immediately followed by retro-orbital injection of saline or 1 × 10⁹ platelets from COX1 deficient mice (COX1^−/−) or wild-type littermates (COX1^+/+) in 0.2 ml saline. Blood was collected before or at 1, 2, 4, and 8 hours after LPS injection. TNF-α (a) and IL-6 (b) concentrations in plasma were measured. Values are means ± s.d. (n = 5). Differences between two groups were assessed using unpaired two-tailed Student’s t-test. (c) C57BL/6 mice received intraperitoneal injection of LPS (10 mg/kg) immediately followed by retro-orbital injection of 1 × 10⁹ platelets from either COX1 knockout mice or wild type littermates in 0.2 ml saline or saline. Survival rate was observed for 3 days. P<0.001 between control group and the group injected with wild type platelets; p>0.2 between control group and the group injected with COX1^−/− platelets by Wald test.(d) C57BL/6 mice received intraperitoneal injection of aspirin (10 or 25 mg/kg). Blood was collected at 30 min after aspirin injection. Washed platelets were incubated with thrombin (0.1 U/ml) for 5 min, and TXB₂ in supernatant was measured by ELISA kit and expressed as relative to platelets from control mice (without aspirin injection) in the absence of thrombin. Values are means ± s.d. (n = 3). Differences between two groups were assessed using unpaired two-tailed Student’s t-test. (e,f) C57BL/6 mice received intraperitoneal injection of aspirin (25 mg/kg) 30 min prior to intraperitoneal injection of LPS (10 mg/kg). Blood was collected before or at 1, 2, 4, and 8 hours after LPS injection. TNF-α (e) and IL-6 (f) concentrations in plasma were measured. Values are means ± s.d. (n = 6). Differences between two groups were assessed using unpaired two-tailed Student’s t-test. (g) C57BL/6 mice received intraperitoneal injection of aspirin (25 mg/kg) at 30 min prior to intraperitoneal injection of LPS (10 mg/kg). Survival rate was observed for 3 days. P value was calculated by Wald test in a discrete time hazard model using version 9.2 of SAS software.