Preconditioning with Hemin Decreases <em>Plasmodium chabaudi adami</em> Parasitemia and Inhibits Erythropoiesis in BALB/c Mice

<div><p>Increased susceptibility to bacterial and viral infections and dysfunctional erythropoiesis are characteristic of malaria and other hemolytic hemoglobinopathies. High concentrations of free heme are common in these conditions but little is known about the effect of heme on adaptive immunity and erythropoiesis. Herein, we investigated the impact of heme (hemin) administration on immune parameters and steady state erythropoiesis in BALB/c mice, and on parasitemia and anemia during <em>Plasmodium chabaudi adami</em> infection. Intra-peritoneal injection of hemin (5 mg/Kg body weight) over three consecutive days decreased the numbers of splenic and bone marrow macrophages, IFN-γ responses to CD3 stimulation and T_h1 differentiation. Our results show that the numbers of erythroid progenitors decreased in the bone marrow and spleen of mice treated with hemin, which correlated with reduced numbers of circulating reticulocytes, without affecting hemoglobin concentrations. Although blunted IFN-γ responses were measured in hemin-preconditioned mice, the mice developed lower parasitemia following <em>P.c.adami</em> infection. Importantly, anemia was exacerbated in hemin-preconditioned mice with malaria despite the reduced parasitemia. Altogether, our data indicate that free heme has dual effects on malaria pathology.</p> </div

Impact of heme on the clearance of <i>Plasmodium</i> infection.

<p><i>P. chabaudi adami</i> DK (10⁵ parasitized RBCs) were inoculated by the intravenous route in PBS (Ctrl)- and hemin (HE)-preconditioned mice (5 mg/kg, for 3 consecutive days) 24 h after the last injection. Parasitemia was followed daily from tail-tip blood smears for estimation of the kinetics of infection (A) and cumulative (B) and peak parasitemia (C). Values represent the mean ± SEM from two independent experiments (n = 8) and were compared using a non-parametric Student <i>t</i> test. **<i>p</i><0.01; ***<i>p</i><0.001.</p

Immune parameters in control and heme-preconditioned BALB/c mice 10 days after <i>P. c. adami</i> infection.

<p>PBS (Ctrl) and hemin (HE)-treated mice were euthanized and spleen was recovered to assess the number of macrophages (F4/80⁺ cells) (A) and CD4 T cells (B). Spleen cells were stimulated with anti-CD3 monoclonal antibody for 48 h to assess IFN-γ (C) and IL-4 (D) production by ELISA. Purified CD4 T cells were stimulated with anti-CD3 and anti-CD28 monoclonal antibodies to estimate the production of IFN-γ (E) and IL-4 (F). Data represent the mean ± SEM from two independent experiments (n = 7–12) and values were compared using a non-parametric Student <i>t</i> test.</p

Alterations in bone and erythropoiesis in hemolytic anemia: comparative study in bled, phenylhydrazine-treated and Plasmodium-infected mice.

Sustained erythropoiesis and concurrent bone marrow hyperplasia are proposed to be responsible for low bone mass density (BMD) in chronic hemolytic pathologies. As impaired erythropoiesis is also frequent in these conditions, we hypothesized that free heme may alter marrow and bone physiology in these disorders. Bone status and bone marrow erythropoiesis were studied in mice with hemolytic anemia (HA) induced by phenylhydrazine (PHZ) or Plasmodium infection and in bled mice. All treatments resulted in lower hemoglobin concentrations, enhanced erythropoiesis in the spleen and reticulocytosis. The anemia was severe in mice with acute hemolysis, which also had elevated levels of free heme and ROS. No major changes in cellularity and erythroid cell numbers occurred in the bone marrow of bled mice, which generated higher numbers of erythroid blast forming units (BFU-E) in response to erythropoietin. In contrast, low numbers of bone marrow erythroid precursors and BFU-E and low concentrations of bone remodelling markers were measured in mice with HA, which also had blunted osteoclastogenesis, in opposition to its enhancement in bled mice. The alterations in bone metabolism were accompanied by reduced trabecular bone volume, enhanced trabecular spacing and lower trabecular numbers in mice with HA. Taken together our data suggests that hemolysis exerts distinct effects to bleeding in the marrow and bone and may contribute to osteoporosis through a mechanism independent of the erythropoietic stress

Evidence of IL-17, IP-10, and IL-10 involvement in multiple-organ dysfunction and IL-17 pathway in acute renal failure associated to Plasmodium falciparum malaria.

International audienceBACKGROUND: Plasmodium falciparum malaria in India is characterized by high rates of severe disease, with multiple organ dysfunction (MOD)-mainly associated with acute renal failure (ARF)-and increased mortality. The objective of this study is to identify cytokine signatures differentiating severe malaria patients with MOD, cerebral malaria (CM), and cerebral malaria with MOD (CM-MOD) in India. We have previously shown that two cytokines clusters differentiated CM from mild malaria in Maharashtra. Hence, we also aimed to determine if these cytokines could discriminate malaria subphenotypes in Odisha.METHODS: P. falciparum malaria patients from the SCB Medical College Cuttack in the Odisha state in India were enrolled along with three sets of controls: healthy individuals, patients with sepsis and encephalitis (n = 222). We determined plasma concentrations of pro- and anti-inflammatory cytokines and chemokines for all individuals using a multiplex assay. We then used an ensemble of statistical analytical methods to ascertain whether particular sets of cytokines/chemokines were predictors of severity or signatures of a disease category.RESULTS: Of the 26 cytokines/chemokines tested, 19 increased significantly during malaria and clearly distinguished malaria patients from controls, as well as sepsis and encephalitis patients. High amounts of IL-17, IP-10, and IL-10 predicted MOD, decreased IL-17 and MIP-1α segregated CM-MOD from MOD, and increased IL-12p40 differentiated CM from CM-MOD. Most severe malaria patients with ARF exhibited high levels of IL-17.CONCLUSION: We report distinct differences in cytokine production correlating with malarial disease severity in Odisha and Maharashtra populations in India. We show that CM, CM-MOD and MOD are clearly distinct malaria-associated pathologies. High amounts of IL-17, IP-10, and IL-10 were predictors of MOD; decreased IL-17 and MIP-1α separated CM-MOD from MOD; and increased IL-12p40 differentiated CM from CM-MOD. Data also suggest that the IL-17 pathway may contribute to malaria pathogenesis via different regulatory mechanisms and may represent an interesting target to mitigate the pathological processes in malaria-associated ARF

Baseline characteristics.

<p>Baseline characteristics.</p

Multiple correlation analyses of [EPO + hemopexin + total heme] with [TNF-α + IL-10 + IP-10 + MCP-1] during MM.

<p>Multiple correlation analyses of [EPO + hemopexin + total heme] with [TNF-α + IL-10 + IP-10 + MCP-1] during MM.</p

Multiple correlation analyses of [EPO + hemopexin + total heme] with [TNF-α + IL-10 + IP-10 + MCP-1] for all <i>P</i>. <i>falciparum</i>-infected patients.

<p>Multiple correlation analyses of [EPO + hemopexin + total heme] with [TNF-α + IL-10 + IP-10 + MCP-1] for all <i>P</i>. <i>falciparum</i>-infected patients.</p