Consistent with their seminal role in detecting infection, both mouse bone marrow-derived and splenic CD11c+ dendritic cells (DCs) exhibited higher levels of uptake of Plasmodium chabaudi-parasitized RBCs (pRBCs) than of noninfected RBCs (nRBCs) as determined by our newly developed flow cytometric technique using the dye CFSE to label RBCs before coculture with DCs. To confirm that expression of CFSE by CD11c+ cells following coculture with CFSE-labeled pRBCs represents internalization of pRBC by DCs, we showed colocalization of CFSE-labeled pRBCs and PE-labeled CD11c+ DCs by confocal fluorescence microscopy. Treatment of DCs with cytochalasin D significantly inhibited the uptake of pRBCs, demonstrating that uptake is an actin-dependent phagocytic process. The uptake of pRBCs by splenic CD11c+ DCs was significantly enhanced after infection in vivo and was associated with the induction of DC maturation, IL-12 production, and stimulation of CD4+ T cell proliferation and IFN-γ production. These results suggest that DCs selectively phagocytose pRBCs and present pRBC-derived Ags to CD4+ T cells, thereby promoting development of protective Th1-dependent immune responses to blood-stage malaria infection.

Low reticulocytosis, indicating reduced red blood cell (RBC) output, is an important feature of severe malarial anemia. Evidence supports a role for Plasmodium products, especially hemozoin (Hz), in suppressed erythropoiesis during malaria, but the mechanism(s) involved remains unclear. Here, we demonstrated that low reticulocytosis and suppressed erythropoietin (Epo)-induced erythropoiesis are features of malarial anemia in Plasmodium yoelii- and Plasmodium berghei ANKA-infected mice, similar to our previous observations in Plasmodium chabaudi AS-infected mice. The magnitude of decreases in RBC was a reflection of parasitemia level, but low reticulocytosis was evident despite differences in parasitemia, clinical manifestation, and infection outcome. Schizont extracts and Hz from P. falciparum and P. yoelii and synthetic Hz suppressed Epo-induced proliferation of erythroid precursors in vitro but did not inhibit RBC maturation. To determine whether Hz contributes to malarial anemia, P. yoelii-derived or synthetic Hz was administered to naive mice, and the development of anemia, reticulocytosis, and RBC turnover was determined. Parasite-derived Hz induced significant decreases in RBC and increased RBC turnover with compensatory reticulocytosis, but anemia was not as severe as that in infected mice. Our findings suggest that parasite factors, including Hz, contribute to severe malarial anemia by suppressing Epo-induced proliferation of erythroid precursors.

Abstract. In December 2004, Togo was the first country to conduct a nationwide free insecticide-treated net (ITN) distribution as part of its National Integrated Child Health Campaign. Community-based cross-sectional surveys were conducted one and nine months post-campaign as part of a multidisciplinary evaluation of the nationwide distribution of ITNs to children 9-59 months of age to evaluate ITN ownership, equity, and use. Our results demonstrated that at one month post-campaign, 93.1% of all eligible children received an ITN. Household ITN ownership and equity increased significantly post-campaign. Nine months post-campaign, 78.6% of households with a child eligible to participate in the campaign retained at least one campaign net. Use by eligible children was 43.5% at one month post-campaign (during the dry season) and 52.9% at nine months post-campaign (during the rainy season). Household ownership of at least one ITN increased from 8.0% pre-campaign to 62.5% one month post-campaign. Together, these findings demonstrate that in this setting, increased household ITN ownership, equity, and retention can be achieved on a national scale through free ITN distribution during an integrated campaign.

The online version of this article contains a supplementary appendix. BackgroundThe contribution of pro-inflammatory cytokines to the pathogenesis of malarial anemia has been studied extensively but the roles of Th2 cytokines remain unknown. Here, we investigated the role of signal transducer and activator of transcription (STAT)6-mediated responses in erythropoietic suppression during acute malaria infection in mice. Design and MethodsNaïve and/or erythropoietin-treated wild-type and STAT6 -/-mice were infected with Plasmodium chabaudi AS (P. chabaudi), and the effects on parasitemia, hematologic parameters, erythropoietin receptor, TER119, and CD71 expression, in vitro erythropoietin-stimulated proliferation of splenic erythroid precursors, and serum cytokine levels were analyzed. To explore the role of interleukin-4 in STAT6-dependent erythropoietic suppression, mice were treated in vivo with a monoclonal antibody to interleukin-4 and the effects on parasitemia, hematologic parameters, and cytokine levels were analyzed. Results Infected STAT6-/-mice developed enhanced reticulocytosis compared to wild-type mice despite higher parasitemia and a similar course of anemia. Enhanced reticulocytosis in infected STAT6 -/-mice was associated with an increased frequency of late-stage erythroblasts, fewer leukocytes expressing CD71, and increased erythropoietin-stimulated proliferation of splenocytes compared to infected wild-type mice. Interleukin-4-depleted wildtype mice had increased levels of parasitemia and a course of reticulocytosis similar to responses observed in infected STAT6 -/-mice. Determination of serum cytokine levels in STAT6 -/-and wild-type mice depleted of interleukin-4 by treatment with mAb revealed significantly lower levels of interferon-γ compared to control wild-type mice during infection. ConclusionsTogether, these findings provide evidence for a STAT6-dependent mechanism in mediating erythropoietic suppression during acute blood-stage malaria and indicate a role for interleukin-4 and possibly interferon-γ in STAT6-induced erythropoietic suppression.Key words: malaria, anemia, erythropoiesis, STAT6, interleukin-4, interferon-γ.Citation: Thawani N, Tam M, and Stevenson MM. STAT6-mediated suppression of erythropoiesis in an experimental model of malarial anemia. Haematologica 2009; 94:195-204. doi:10.3324/haematol.13422 ©2009 Ferrata Storti Foundation. This is an open-access paper. STAT6-mediated suppression of erythropoiesis in an experimental model of malarial anemia ABSTRACT© F e r r a t a S t o r t i F o u n d a t i o n N. Thawani et al.

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