Plasmodium products contribute to severe malarial anemia by inhibiting erythropoietin-induced proliferation of erythroid precursors.

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.

STAT6-mediated suppression of erythropoiesis in an experimental model of malarial anemia.

BACKGROUND: The 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 METHODS: Naïve and/or erythropoietin-treated wild-type and STAT6(-/-) mice were infected with Plasmodium chabaudi AS (P. chabaudi), and the effects 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 wild-type 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-gamma compared to control wild-type mice during infection. CONCLUSIONS: Together, 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-gammain STAT6-induced erythropoietic suppression.

Interferon-gamma mediates suppression of erythropoiesis but not reduced red cell survival following CpG-ODN administration in vivo.

OBJECTIVE: Cytokines released during inflammatory processes have been proposed to play a central role in mediating mechanism(s) leading to anemia. Here, we used CpG-ODN to investigate the effects of a pro-inflammatory response on the pathophysiological processes leading to anemia. METHODS: Naïve and erythropoietin (EPO)-treated mice were injected for 2 days with 100 microg CpG-ODN or control ODN and the effects on the course of red blood cell (RBC) and reticulocyte counts, RBC turnover, and EPO-stimulated maturation of erythroid cells were analyzed. To study the effect of CpG-ODN on erythroid cell maturation in vitro, we obtained primary EPO-responsive cells by treating mice with thiamphenicol (15 mg/g body weight). RESULTS: CpG-ODN-treated mice developed anemia, which persisted for 5 days and was associated with a 50% reduction in EPO-stimulated differentiation of EPOR+ cells to TER119+ erythroblasts. CpG-ODN-induced suppression required accessory cells, including antigen presenting cells, which activated other cells to produce pro-inflammatory cytokines. In vitro neutralization of IFN-gamma, but not IL-12, TNF-alpha, IFN-alpha, IL-1alpha, or IL-1beta, abrogated the erythropoietic suppression induced by CpG-ODN. The anemia observed in CpG-ODN-treated mice was also associated with reduced RBC survival in vivo, as demonstrated by a sevenfold to eightfold higher turnover of biotinylated RBC compared to control ODN-treated mice. In vivo IFN-gamma neutralization confirmed that IFN-gamma contributed to erythropoietic suppression but not reduced RBC survival. CONCLUSIONS: Together, these results demonstrate that CpG-ODN anemia is associated with suppressed erythropoiesis and decreased RBC survival. Importantly, CpG-ODN-induced IFN-gamma was found to be the major factor mediating erythropoietic suppression but not decreased RBC survival.

Rapid scale-up of long-lasting insecticide-treated bed nets through integration into the national immunization program during child health week in Togo, 2004.

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.

Factors associated with coverage and usage of long-lasting insecticidal nets in madagascar.

In October 2007, long-lasting insecticidal nets (LLINs) were distributed in 59 of the 111 districts in Madagascar as part of a nationwide child survival campaign. A community-based cross-sectional survey was conducted six months post-campaign to evaluate net ownership, use and equity. Here, we examined the effects of socioeconomic factors on LLIN ownership and usage in districts with and without net distribution during the campaign. Our data demonstrated that in districts with LLIN distribution, LLIN ownership was similar across all wealth groups in households with at least one child under the age of five years (90.5% versus 88.6%); in districts without net distribution, 57.8% of households in the poorest tertile compared to 90.1% of households in the least poor tertile owned at least one LLIN. In contrast, in LLIN-owning households, both in districts with and without net distribution, higher socio-economic status was not associated with use among children under five years. These findings suggest that socio-economic status contributes to the household net ownership but once a household owns a net, socio-economic status is not associated with net use.

Interaction of mouse dendritic cells and malaria-infected erythrocytes: uptake, maturation, and antigen presentation.

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-gamma 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.