Platelet induction of the acute-phase response is protective in murine experimental cerebral malaria

Abstract

Platelets are most recognized as the cellular mediator of thrombosis, but they are increasingly appreciated for their immunomodulatory roles, including responses to Plasmodium infection. Platelet interactions with endothelial cells and leukocytes contribute significantly to the pathogenesis of experimental cerebral malaria (ECM). Recently, it has been suggested that platelets not only have an adverse role in cerebral malaria, but platelets may also be protective in animal models of uncomplicated malaria. We now demonstrate that these diverse and seemingly contradictory roles for platelets extend to cerebral malaria models and are dependent on the timing of platelet activation during infection. Our data show that platelets are activated very early in ECM and have a central role in initiation of the acute-phase response to blood-stage infection. Unlike platelet depletion or inhibition postinfection, preinfection platelet depletion or treatment with a platelet inhibitor is not protective. Additionally, we show that platelet-driven acute-phase responses have a major role in protecting mice from ECM by limiting parasite growth. Our data now suggest that platelets have a complex role in ECM pathogenesis: platelets help limit parasite growth early postinfection, but with continued platelet activation as the disease progresses, platelets contribute to ECM-associated inflammation.

Figure 1. The Role of Platelets in ECM is Time Dependent

A. Platelets are activated early in ECM. Mice were infected with P. berghei and plasma PF4 concentration measured by ELISA (bars). The corresponding parasitemia (line) was also determined at the same time points. (N=5 per timepoint, mean ± SD,*P<0.05). B. Platelet depletion pre-infection is not ECM protective. Mice were platelet depleted 24 hrs before or after P. berghei ANKA infection and survival monitored Mice depleted of platelets post infection, but not pre-infection had improved survival. (N=5,*P<0.05, log-rank test). C. Platelet inhibition pre-infection is also not protective. Mice were treated with clopidogrel beginning 24 hours pre- or post P. berghei infection and survival monitored. (N=10,* P<0.05 log-rank test). D. Mice platelet depleted pre-infection have in increased parasitemia. Mice were platelet depleted 24 hours prior to P. bergehi infection and the number of infected RBCs compared with non platelet-depleted controls was determined at day 5 (N=5, mean ± S.D, *P≤0.05).

Figure 2. Platelets drive the acute phase response to P. berghei infection

A. Mice were platelet-depleted or treated with control IgG 24 hrs pre-infection and plasma SAP measured by ELISA (N=5, mean ± S.D.*P<0.05). B. Mice were platelet-depleted or treated with IgG 24 hrs pre-infection and plasma serum amyloid A (SAA) measured by ELISA (n=5, mean ± S.D.*P<0.03). C. Mice were platelet-depleted or treated with IgG 24 hours post-infection and plasma serum amyloid P (SAP) measured by ELISA (n=5, mean ± S.D). D. Mice were platelet-depleted or treated with IgG 24 hours post-infection and plasma serum amyloid A (SAA) measured by ELISA (n=5, mean ± S.D.*P<0.03).

Figure 3. Platelets are a major source of IL-1β

A. Platelets are a primary mediator of IL-1β during the acute phase response to ECM. Mice were platelet depleted or IgG treated before infection and plasma IL1-β measured by ELISA (N=5, mean ± SD,*P≤ 0.01). B. Plasma IL-1β in post-infection platelet depleted mice. Mice were platelet depleted or control IgG treated 24 hours post-infection and plasma IL1-β measured by ELISA (N=5, mean ± SD). C. IL-1β reconstitution restores APP production in pre-infection platelet depleted mice. Mice were platelet depleted or treated with control IgG 24 hrs pre-infection; platelet depleted mice were treated with control buffer (PBS) or mrIL-1β at the time of infection, 24 and 48 hrs post infection. Plasma SAP levels were measured by ELISA (N=5, mean ± SD, *P<0.05). D. Platelets from P. berghei infected mice have increased IL-1β. Mice were infected with P. berghei, platelets isolated and intracellular IL-1β analyzed by flow cytometry (N=5, mean ± SD *P≤0.05).

Figure 4. Platelets are recruited to the liver early post P berghei infection

A. Platelets are recruited to the liver post-infection. 24 hrs after infection mice were injected with anti-platelet fluorescent antibody and sacrificed. Livers were imaged with a fluorescent microscope (Representative Images). B. Platelet foci were quantified from 10 different fields. (N=5, mean ± S.D., *P≤0.01, unpaired Student’s t-test). C. Platelets (P) interact with liver sinusoidal endothelial cells (LSEC) during infection (representative TEM images). Ultrathin liver sections from uninfected controls (left) and infected mice (right) 24 hrs post P berghei infection.

Figure 5. The Acute Phase Response is Protective in ECM

A. Exogenous SAP administration of platelet depleted P. berghei infected mice returns them to control survival levels. Mice were platelet depleted 24 hours pre-infection or platelet depleted and treated with rSAP for the first 3 days post infection. Survival (N=5, *P<0.05 log-rank test vs platelet depleted+SAP). B. CRP-Tg mice are protected from ECM. CRP-Tg or WT mice were infected with P. berghei and survival monitored (N=5,*P<0.05 log-rank test). C. CRP reduces the parasite burden in ECM. Mice were infected with P. berghei and parasitemia determined by flow cytometry analysis of SybrGreen labeled iRBCs (N=5, *P<0.05 unpaired Students t-test). D. CRP reduces the parasite burden in P yoelii infections. Mice were infected with P yoelii and parasitemia determined by flow cytometry analysis of SyBrGreen stained iRBCs (N=8, mean ± SD, *P<0.05, unpaired Students t-test).

Figure 6. Platelet Mediated Acute Phase Response in ECM

Platelets are activated by infected RBCs and in an IL-1β dependent manner induce the production of acute phase proteins such as CRP and SAP. Acute phase proteins decrease parasitemia and mediate survival in ECM.