Quantification of host-mediated parasite clearance during blood-stage Plasmodium infection and anti-malarial drug treatment in mice

A major mechanism of host-mediated control of blood-stage Plasmodium infection is thought to be removal of parasitized red blood cells (pRBCs) from circulation by the spleen or phagocytic system. The rate of parasite removal is thought to be further increased by anti-malarial drug treatment, contributing to the effectiveness of drug therapy. It is difficult to directly compare pRBC removal rates in the presence and absence of treatment, since in the absence of treatment the removal rate of parasites is obscured by the extent of ongoing parasite proliferation. Here, we transfused a single generation of fluorescently-labelled Plasmodium berghei pRBCs into mice, and monitored both their disappearance from circulation, and their replication to produce the next generation of pRBCs. In conjunction with a new mathematical model, we directly estimated host removal of pRBCs during ongoing infection, and after drug treatment. In untreated mice, pRBCs were removed from circulation with a half-life of 15.1 h. Treatment with various doses of mefloquine/artesunate did not alter the pRBC removal rate, despite blocking parasite replication effectively. An exception was high dose artesunate, which doubled the rate of pRBC removal (half-life of 9.1 h). Phagocyte depletion using clodronate liposomes approximately halved the pRBC removal rate during untreated infection, indicating a role for phagocytes in clearance. We next assessed the importance of pRBC clearance for the decrease in the parasite multiplication rate after high dose artesunate treatment. High dose artesunate decreased parasite replication ∼46-fold compared with saline controls, with inhibition of replication contributing 23-fold of this, and increased pRBC clearance contributing only a further 2.0-fold. Thus, in our in vivo systems, drugs acted primarily by inhibiting parasite replication, with drug-induced increases in pRBC clearance making only minor contributions to overall drug effect

IFN regulatory factor 3 balances Th1 and T follicular helper immunity during nonlethal blood-stage Plasmodium infection

Differentiation of CD4Th cells is critical for immunity to malaria. Several innate immune signaling pathways have been implicated in the detection of blood-stageparasites, yet their influence over Th cell immunity remains unclear. In this study, we usedreactive TCR transgenic CD4T cells, termed PbTII cells, during nonlethalAS and17XNL infection in mice, to examine Th cell development in vivo. We found no role for caspase1/11, stimulator of IFN genes, or mitochondrial antiviral-signaling protein, and only modest roles for MyD88 and TRIF-dependent signaling in controlling PbTII cell expansion. In contrast, IFN regulatory factor 3 (IRF3) was important for supporting PbTII expansion, promoting Th1 over T follicular helper (Tfh) differentiation, and controlling parasites during the first week of infection. IRF3 was not required for early priming by conventional dendritic cells, but was essential for promoting CXCL9 and MHC class II expression by inflammatory monocytes that supported PbTII responses in the spleen. Thereafter, IRF3-deficiency boosted Tfh responses, germinal center B cell and memory B cell development, parasite-specific Ab production, and resolution of infection. We also noted a B cell-intrinsic role for IRF3 in regulating humoral immune responses. Thus, we revealed roles for IRF3 in balancing Th1- and Tfh-dependent immunity during nonlethal infection with blood-stageparasites