Platelet factor 4 mediates inflammation in experimental cerebral malaria

Abstract

Cerebral malaria (CM) is a major complication of Plasmodium falciparum infection in children. The pathogenesis of CM involves vascular inflammation, immune stimulation, and obstruction of cerebral capillaries. Platelets have a prominent role in both immune responses and vascular obstruction. We now demonstrate that the platelet-derived chemokine, platelet factor 4 (PF4)/CXCL4, promotes the development of experimental cerebral malaria (ECM). Plasmodium-infected red blood cells (RBCs) activated platelets independently of vascular effects, resulting in increased plasma PF4. PF4 or chemokine receptor CXCR3 null mice had less severe ECM, including decreased T cell recruitment to the brain, and platelet depletion or aspirin treatment reduced the development of ECM. We conclude that Plasmodium-infected RBCs can directly activate platelets, and platelet-derived PF4 then contributes to immune activation and T cell trafficking as part of the pathogenesis of ECM.

Figure 1. Platelets are activated by Plasmodium infected RBC

(A) P. falciparum infected RBC increase platelet activation. GPIIb/IIIa. Human platelets were incubated with buffer, control RBC, or PfRBC for 30 mins and platelet activation measured by GPIIb/IIIa receptor activation (PAC-1 antibody) using flow cytometry. TRAP (5 µM) treated platelets were used as positive controls (n=4, mean ± S.D. *P<0.01). (B) P-selectin. Human platelets were incubated with control RBC, or PfRBC and surface P-selectin determined using flow cytometry (n=4, mean ± S.D. *P<0.03). (C) PF4 Release. Human platelets were incubated with buffer solution, control RBC, or PfRBC for 30 mins and supernatant PF4 measured by ELISA (n=4, mean ± S.D. *P<0.01). (D) P. falciparum infected RBC membranes activate platelets. Control or PfRBC red blood cell ghosts were prepared and incubated with platelets. Platelet activation was determined by FACS for PAC-1 binding (n=4, mean ± S.D. *P<0.01). (E) PfRBC activates platelets via CD36. Human platelets were incubated with control RBC or PfRBC in the presence of control IgG or CD36 blocking antibody Fab fragments for 30 mins and platelet activation determined by FACS (n=4, mean ± S.D. *P<0.05).

Figure 2. Platelets are activated in ECM

(A) Cerebral thrombi in ECM. P. berghei infected mice have cerebral vascular inflammation and hemorrhage (arrowheads top) and vWf positive microvascular thrombi (arrows bottom). (B) P. berghei infected mice have increased brain PF4. Brains were harvested from control and malaria infected mice on day 5 p.i. PF4 concentration was determined in brain lysates by ELISA (n=5 mean ± S.D. *P<0.01 vs control). (C) Malaria infected mice have increased circulating activated platelets. Platelets from P. berghei infected mice were isolated on day 2 and 4 post infection, surface P-selectin and fibrinogen binding determined by flow cytometry and expressed as change in fluorescence vs control platelets (n=5, mean ± S.D. *P<0.01 vs Control). (D) P. berghei infected mice have increased plasma PF4. Plasma from infected and control mice was isolated and PF4 concentration determined by ELISA (n=5 pre-infection and Day 4, n=3 day 6 mean ± S.D. *P<0.01 vs pre-infection).

Figure 3. PF4 drives the development of ECM

(A) PF4 increases ECM. WT, PF4^−/−, CXCR3^−/−, and PF4 reconstituted PF4^−/− mice were infected with P. berghei and monitored for death (*P<0.01, log-rank test). (B) WT and PF4^−/− P. berghei infected mice have equal parasite burden (n=5 days 1–5, n=3 day 7, mean ± S.D.). (C) BBB integrity is protected in PF4^−/− and CXCR3^−/− mice. On day 5 p.i., P. berghei infected and control mice were injected with 200 µL of 2% Evans Blue Dye IV and dye extravasation determined (n=5 mean ± S.D., *P<0.05 vs WT). (D) P. berghei infected PF4^−/− and CXCR3^−/− mice have decreased cerebral vascular damage compared to WT infected mice. H&E top, vWf immunohistochemistry bottom. (E) Platelet inhibition decreases ECM. P. berghei infected mice were treated with ASA, Plavix/clopidogrel, or platelet depleted (*P<0.01, log-rank test).

Figure 4. PF4 Promotes Immune Stimulation and T-Cell Trafficking in ECM

(A and B) Plasma TNFα and IFNγ are not increased in infected PF4^−/− mice. Plasma was isolated from control or P. berghei infected mice on day 5 p.i. and (A) TNFα and (B) IFNγ concentrations determined by ELISA (n=5, *P<0.01 vs WT control). (C) PF4 increases macrophage TNFα production. Peritoneal macrophages were incubated with 1 µg of PF4 for 48 hrs and TNFα measured in the culture supernatant by ELISA (n=4, mean ± S.D., *P<0.01 vs Control). (D) PF4 increases T-cell TNFα. TNFα was measured in splenocyte culture supernatants by ELISA (n=3–6, mean ± S.D., *P<0.01 vs Control). (E) PF4 increases the number of CXCR3 positive T-cells in vivo. T-cells were isolated from the spleens of P. berghei infected mice on day 5 p.i. CXCR3 expression was determined by FACS (n=5 mean ± S.D., mean ± S.D., *P<0.01 vs WT control). (F) PF4 directly increases T-cell CXCR3 expression. Splenocytes were incubated in anti-CD3 coated wells with anti-CD28 antibody in the presence of control or 1 µg/mL of recombinant PF4 for 4 days. CXCR3 surface expression was determined by FACS (n=4, mean ± S.D., *P<0.05 vs Control). (G) PF4 signaling increases T-cell brain infiltration. Brains were isolated and CD8+ and CD4+ T-cells determined by FACS (n=4–5, mean ± S.D., *P<0.05 vs WT Control). (H) Aspirin treated mice have fewer T-cell infiltrates when P. berghei infected as compared to control infected mice (n=5, mean ± S.D., *P<0.02 vs WT Control).