Multifaceted Role of Heme during Severe Plasmodium falciparum Infections in India

Several immunomodulatory factors are involved in malaria pathogenesis. Among them, heme has been shown to play a role in the pathophysiology of severe malaria in rodents, but its role in human severe malaria remains unclear. Circulating levels of total heme and its main scavenger, hemopexin, along with cytokine/chemokine levels and biological parameters, including hemoglobin and creatinine levels, as well as transaminase activities, were measured in the plasma of 237 Plasmodium falciparum-infected patients living in the state of Odisha, India, where malaria is endemic. All patients were categorized into well-defined groups of mild malaria, cerebral malaria (CM), or severe noncerebral malaria, which included acute renal failure (ARF) and hepatopathy. Our results show a significant increase in total plasma heme levels with malaria severity, especially for CM and malarial ARF. Spearman rank correlation and canonical correlation analyses have shown a correlation between total heme, hemopexin, interleukin-10, tumor necrosis factor alpha, gamma interferon-induced protein 10 (IP-10), and monocyte chemotactic protein 1 (MCP-1) levels. In addition, canonical correlations revealed that heme, along with IP-10, was associated with the CM pathophysiology, whereas both IP-10 and MCP-1 together with heme discriminated ARF. Altogether, our data indicate that heme, in association with cytokines and chemokines, is involved in the pathophysiology of both CM and ARF but through different mechanisms.Indo-French Centre for the Promotion of Advanced Research, Associated International Laboratory Systems (LIA; CNRS), Immunology and Genetics of Infectious Diseases (SIGID), Department of Biotechnology from the Ministry of Science and Technology of India (DBT), Tata Institute of Fundamental Research (TIFR) (intramural funds), Université Lille (doctoral contract), IFCPAR (Raman-Charpak award), College Doctoral Lille Nord de France (AAP n10 award), Fondation des Treille, Conseil Régional du Nord-Pas de Calais

The modulatory effect of heme on the response of T lymphocytes and glial cells : and its impact on the pathophysiology of severe malaria

La phase érythrocytaire du cycle de Plasmodium est caractérisée par une hémolyse importante et la libération d’hème chez l’hôte. Grâce à ses activités pro-oxydantes et immunomodulatrices, l’hème contribuerait au dysfonctionnement de la réponse immune observée durant le paludisme grave. Ainsi, ce travail de thèse avait pour but de clarifier le rôle de l’hème sur la réponse des lymphocytes T et des cellules gliales au cours du neuropaludisme et de l’anémie palustre sévère dans des modèles murins de paludisme. De plus, nous avons étendu nos études au paludisme chez des patients infectés par P. falciparum manifestant différents phénotypes cliniques. D‘après nos résultats l’hème diminue la réponse délétère de type 1 des lymphocytes T CD4 et des microglies chez les modèles murins. Alors que cette réponse est essentielle à la clairance du parasite, le prétraitement des souris BALB/c avec l’hème induit une parasitémie plus faible, en comparaison au groupe non traité, qui est également associée à une anémie liée à une dysérythropoïèse. Cependant, l’injection d’hème chez des souris C57BL/6 pendant l’infection les protège du neuropaludisme en diminuant le recrutement des lymphocytes T, la réponse de type M1 des microglies et la production de cytokines pro-inflammatoires dans le cerveau. Par l’étude réalisée dans l’Odisha en Inde nous avons corroboré l’ensemble de ces résultats avec des données cliniques, et montrons que les niveaux plasmatiques d’hème augmentent chez les patients atteints de neuropaludisme et corrèlent positivement avec les cytokines IP-10, IL-10, TNF-alpha et MCP-1. Nous soulignons dans notre étude la complexité de la physiopathologie du paludisme grave.During the erythrocytic phase of the parasite Plasmodium, large amounts of heme are release in circulation. Due to its pro-oxidant, and immunomodulating activities, heme might be responsible for the alterations of the immune response that were reported during severe malaria. Thus, the objectives of this work were to clarify the impact of heme on the response of T lymphocytes and glial cells in rodent models of severe malarial anemia, and cerebral malaria. In addition, we have also looked at the relationship between heme levels and malaria severity in a human cohort of P. falciparum-infected patients manifesting mild and severe malaria. Heme decreased the pro-inflammatory type 1 response of CD4 T lymphocytes and microglia cells in rodent models. Despite the essential role of this response for the elimination of the parasite, BALB/c mice that were preconditioned with heme before infection had lower parasitemia compared to the untreated group. However, heme induced stronger anemia during the infection that was concomitant with alterations in erythropoiesis. In addition, heme injection during infection protected partly C57BL/6 mice from developing cerebral malaria by preventing T cell recruitment to the brain, the M1 response of microglia cells, and the production of pro-inflammatory cytokines by brain cells. Interestingly, we show from our field study in Odisha, India, that plasma heme levels increased with the severity of malaria, and more specifically during cerebral malaria, and correlated positively with the plasma levels of four cytokines: IP-10, IL-10, TNF-alpha and MCP-1. Herein we underscore the complexity of the pathophysiology of severe malaria

Le Neuropaludisme (quand l'hème participe à la forme grave de l'infection par Plasmodium)

Le paludisme est une maladie infectieuse provoquée par des protozoaires du genre Plasmodium. Les signes cliniques associés à l'infection se déclinent sous différents aspects, le neuropaludisme étant la forme la plus grave. Dans les pays à forte transmission palustre, le neuropaludisme atteint majoritairement les enfants de moins de 5 ans et est caractérisé par des convulsions, un coma, et la mort de l'hôte dans près de 18% des cas. Au cours de l'infection palustre, les signes cliniques apparaissent lors de la phase érythrocytaire du cycle du parasite, car en se multipliant dans les globules rouges, ce dernier induit la libération systémique de molécules pro-inflammatoires, dont l'hème. Les excès de cette protoporphyrine ferrique en circulation sont associés à des maladies inflammatoires, comme l'arthérosclérose, ou à des atteintes neurodégénératives apparaissant à la suite d'accidents vasculaires cérébraux. Plusieurs données suggèrent une implication de l'hème dans les différentes étapes clés liées au développement du neuropaludisme. Dans un premier temps, cette protoporphyrine favoriserait l'obstruction mécanique des capillaires cérébraux, et induirait la rupture de la barrière hémato-encéphalique. La conséquence de ce phénomène serait l'activation des cellules du parenchyme cérébral, et la modification de leur réponse vers un profil pro-inflammatoire et pro-apoptotique. Dans un deuxième temps, l'hème participerait à l'induction d'une réponse immune excessive et non adaptée en favorisant l'activation des lymphocytes T et en participant à l'élaboration d'une réponse auto-immune initiée par les lymphocytes B. Pour conclure, au cours du neuropaludisme avéré, le traitement des individus avec des "inhibiteurs" de l'hème pourrait être une association thérapeutique à considérer.CHATENAY M.-PARIS 11-BU Pharma. (920192101) / SudocSudocFranceF

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

Bone marrow and spleen erythroid parameters.

<p>Control (Ctrl) and hemin (HE)-treated mice were euthanized 24 h after the last injection with PBS or HE. Populations of erythroid cells were analyzed in the bone marrow (A) and in the spleen (B) by staining cells with anti-CD71-FITC and anti-Ter119-PE antibodies. The percentage of reticulocytes (CD71⁺ cells) (C) and hemoglobin levels were measured in the blood (D). Macrophages (F4-80⁺ cells) were quantified in femoral bone marrow cell suspensions (E) by staining with anti-F4/80-PE antibody. Erythropoietin (EPO) was measured in the plasma by ELISA (F). Data are mean ± SEM from two independent experiments (n = 4–11 mice per group) and values were compared using a non-parametric Student <i>t</i> test. <i>*p<</i>0.05; **<i>p</i><0.01; ***P<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

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

Erythropoietin Levels Increase during Cerebral Malaria and Correlate with Heme, Interleukin-10 and Tumor Necrosis Factor-Alpha in India.

Cerebral malaria (CM) caused by Plasmodium falciparum parasites often leads to the death of infected patients or to persisting neurological sequelae despite anti-parasitic treatments. Erythropoietin (EPO) was recently suggested as a potential adjunctive treatment for CM. However diverging results were obtained in patients from Sub-Saharan countries infected with P. falciparum. In this study, we measured EPO levels in the plasma of well-defined groups of P. falciparum-infected patients, from the state of Odisha in India, with mild malaria (MM), CM, or severe non-CM (NCM). EPO levels were then correlated with biological parameters, including parasite biomass, heme, tumor necrosis factor (TNF)-α, interleukin (IL)-10, interferon gamma-induced protein (IP)-10, and monocyte chemoattractant protein (MCP)-1 plasma concentrations by Spearman's rank and multiple correlation analyses. We found a significant increase in EPO levels with malaria severity degree, and more specifically during fatal CM. In addition, EPO levels were also found correlated positively with heme, TNF-α, IL-10, IP-10 and MCP-1 during CM. We also found a significant multivariate correlation between EPO, TNF-α, IL-10, IP-10 MCP-1 and heme, suggesting an association of EPO with a network of immune factors in CM patients. The contradictory levels of circulating EPO reported in CM patients in India when compared to Africa highlights the need for the optimization of adjunctive treatments according to the targeted population

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