Ana Pamplona

Heme oxygenase-1 and carbon monoxide suppress the pathogenesis of experimental cerebral malaria

Cerebral malaria claims more than 1 million lives per year. We report that heme oxygenase-1 (HO-1, encoded by Hmox1) prevents the development of experimental cerebral malaria (ECM). BALB/c mice infected with Plasmodium berghei ANKA upregulated HO-1 expression and activity and did not develop ECM. Deletion of Hmox1 and inhibition of HO activity increased ECM incidence to 83% and 78%, respectively. HO-1 upregulation was lower in infected C57BL/6 compared to BALB/c mice, and all infected C57BL/6 mice developed ECM (100% incidence). Pharmacological induction of HO-1 and exposure to the end-product of HO-1 activity, carbon monoxide (CO), reduced ECM incidence in C57BL/6 mice to 10% and 0%, respectively. Whereas neither HO-1 nor CO affected parasitemia, both prevented blood-brain barrier (BBB) disruption, brain microvasculature congestion and neuroinflammation, including CD8+ T-cell brain sequestration. These effects were mediated by the binding of CO to hemoglobin, preventing hemoglobin oxidation and the generation of free heme, a molecule that triggers ECM pathogenesis.

Accumulation of Plasmodium berghei-infected red blood cells in the brain is crucial for the development of cerebral malaria in mice.

ABSTRACT Cerebral malaria is the most severe complication of human infection with Plasmodium falciparum. It was shown that Plasmodium berghei ANKA-induced cerebral malaria was prevented in 100% of mice depleted of CD8+ T cells 1 day prior to the development of neurological signs. However, the importance of parasites in the brains of these mice was never clearly investigated. Moreover, the relevance of this model to human cerebral malaria has been questioned many times, especially concerning the relative importance of leukocytes versus parasitized erythrocytes sequestered in the brain. Here, we show that mice protected from cerebral malaria by CD8+ T-cell depletion have significantly fewer parasites in the brain. Treatment of infected mice with an antimalarial drug 15 to 20 h prior to the estimated time of death also protected mice from cerebral malaria without altering the number of CD8+ T cells in the brain. These mice subsequently developed cerebral malaria with parasitized red blood cells in the brain. Our results clearly demonstrated that sequestration of CD8+ T cells in the brain is not sufficient for the development of cerebral malaria in C57BL/6 mice but that the concomitant presence of parasitized red blood cells is crucial for the onset of pathology. Importantly, these results also demonstrated that the experimental cerebral malaria model shares many features with human pathology and might be a relevant model to study its pathogenesis.

Heme Oxygenase-1 Is an Anti-Inflammatory Host Factor that Promotes Murine Plasmodium Liver Infection

The clinically silent Plasmodium liver stage is an obligatory step in the establishment of malaria infection and disease. We report here that expression of heme oxygenase-1 (HO-1, encoded by Hmox1) is upregulated in the liver following infection by Plasmodium berghei and Plasmodium yoelii sporozoites. HO-1 overexpression in the liver leads to a proportional increase in parasite liver load, and treatment of mice with carbon monoxide and with biliverdin, each an enzymatic product of HO-1, also increases parasite liver load. Conversely, mice lacking Hmox1 completely resolve the infection. In the absence of HO-1, the levels of inflammatory cytokines involved in the control of liver infection are increased. These findings suggest that, while stimulating inflammation, the liver stage of Plasmodium also induces HO-1 expression, which modulates the host inflammatory response, protecting the infected hepatocytes and promoting the liver stage of infection.

VEGF promotes malaria-associated acute lung injury in mice.

The spectrum of the clinical presentation and severity of malaria infections is broad, ranging from uncomplicated febrile illness to severe forms of disease such as cerebral malaria (CM), acute lung injury (ALI), acute respiratory distress syndrome (ARDS), pregnancy-associated malaria (PAM) or severe anemia (SA). Rodent models that mimic human CM, PAM and SA syndromes have been established. Here, we show that DBA/2 mice infected with P. berghei ANKA constitute a new model for malaria-associated ALI. Up to 60% of the mice showed dyspnea, airway obstruction and hypoxemia and died between days 7 and 12 post-infection. The most common pathological findings were pleural effusion, pulmonary hemorrhage and edema, consistent with increased lung vessel permeability, while the blood-brain barrier was intact. Malaria-associated ALI correlated with high levels of circulating VEGF, produced de novo in the spleen, and its blockage led to protection of mice from this syndrome. In addition, either splenectomization or administration of the anti-inflammatory molecule carbon monoxide led to a significant reduction in the levels of sera VEGF and to protection from ALI. The similarities between the physiopathological lesions described here and the ones occurring in humans, as well as the demonstration that VEGF is a critical host factor in the onset of malaria-associated ALI in mice, not only offers important mechanistic insights into the processes underlying the pathology related with malaria but may also pave the way for interventional studies.

A novel carbon monoxide-releasing molecule fully protects mice from severe malaria.

ABSTRACT Severe forms of malaria infection, such as cerebral malaria (CM) and acute lung injury (ALI), are mainly caused by the apicomplexan parasite Plasmodium falciparum. Primary therapy with quinine or artemisinin derivatives is generally effective in controlling P. falciparum parasitemia, but mortality from CM and other forms of severe malaria remains unacceptably high. Herein, we report the design and synthesis of a novel carbon monoxide-releasing molecule (CO-RM; ALF492) that fully protects mice against experimental CM (ECM) and ALI. ALF492 enables controlled CO delivery in vivo without affecting oxygen transport by hemoglobin, the major limitation in CO inhalation therapy. The protective effect is CO dependent and induces the expression of heme oxygenase-1, which contributes to the observed protection. Importantly, when used in combination with the antimalarial drug artesunate, ALF492 is an effective adjunctive and adjuvant treatment for ECM, conferring protection after the onset of severe disease. This study paves the way for the potential use of CO-RMs, such as ALF492, as adjunctive/adjuvant treatment in severe forms of malaria infection.

Cerebral malaria and the hemolysis/methemoglobin/heme hypothesis: Shedding new light on an old disease

Malaria causes more than 1 million deaths every year with cerebral malaria (CM) being a major cause of death in Sub-Saharan African children. The nature of the malaria-associated pathogenesis is complex and multi-factorial. A unified hypothesis involving sequestration of infected red blood cells, systemic host inflammatory response and hemostasis dysfunction has been proposed to explain the genesis of CM. In this review, we discuss the role of hemolysis, methemoglobin and free heme in CM, brought to light by our recent studies in mice as well as by other studies in humans.

B7–CD28 Costimulatory Signals Control the Survival and Proliferation of Murine and Human γδ T Cells via IL-2 Production

γδ T cells play key nonredundant roles in immunity to infections and tumors. Thus, it is critical to understand the molecular mechanisms responsible for γδ T cell activation and expansion in vivo. In striking contrast to their αβ counterparts, the costimulation requirements of γδ T cells remain poorly understood. Having previously described a role for the TNFR superfamily member CD27, we since screened for other nonredundant costimulatory receptors in γδ T cell activation. We report in this article that the Ig superfamily receptor CD28 (but not its related protein ICOS) is expressed on freshly isolated lymphoid γδ T cells and synergizes with the TCR to induce autocrine IL-2 production that promotes γδ cell survival and proliferation in both mice and humans. Specific gain-of-function and loss-of-function experiments demonstrated a nonredundant function for CD28 interactions with its B7 ligands, B7.1 (CD80) and B7.2 (CD86), both in vitro and in vivo. Thus, γδ cell proliferation was significantly enhanced by CD28 receptor agonists but abrogated by B7 Ab-mediated blockade. Furthermore, γδ cell expansion following Plasmodium infection was severely impaired in mice genetically deficient for CD28. This resulted in the failure to mount both IFN-γ–mediated and IL-17–mediated γδ cell responses, which contrasted with the selective effect of CD27 on IFN-γ–producing γδ cells. Our data collectively show that CD28 signals are required for IL-2–mediated survival and proliferation of both CD27+ and CD27− γδ T cell subsets, thus providing new mechanistic insight for their modulation in disease models.

TCR signal strength controls thymic differentiation of discrete proinflammatory [gamma][delta] T cell subsets

The mouse thymus produces discrete γδ T cell subsets that make either interferon-γ (IFN-γ) or interleukin 17 (IL-17), but the role of the T cell antigen receptor (TCR) in this developmental process remains controversial. Here we show that Cd3g+/− Cd3d+/− (CD3 double-haploinsufficient (CD3DH)) mice have reduced TCR expression and signaling strength on γδ T cells. CD3DH mice had normal numbers and phenotypes of αβ thymocyte subsets, but impaired differentiation of fetal Vγ6+ (but not Vγ4+) IL-17-producing γδ T cells and a marked depletion of IFN-γ-producing CD122+ NK1.1+ γδ T cells throughout ontogeny. Adult CD3DH mice showed reduced peripheral IFN-γ+ γδ T cells and were resistant to experimental cerebral malaria. Thus, TCR signal strength within specific thymic developmental windows is a major determinant of the generation of proinflammatory γδ T cell subsets and their impact on pathophysiology.

Simple flow cytometric detection of haemozoin containing leukocytes and erythrocytes for research on diagnosis, immunology and drug sensitivity testing

BackgroundMalaria pigment (haemozoin, Hz) has been the focus of diverse research efforts. However, identification of Hz-containing leukocytes or parasitized erythrocytes is usually based on microscopy, with inherent limitations. Flow cytometric detection of depolarized Side-Scatter is more accurate and its adaptation to common bench top flow cytometers might allow several applications. These can range from the ex-vivo and in-vitro detection and functional analysis of Hz-containing leukocytes to the detection of parasitized Red-Blood-Cells (pRBCs) to assess antimalarial activity.MethodsA standard benchtop flow cytometer was adapted to detect depolarized Side-Scatter. Synthetic and Plasmodium falciparum Hz were incubated with whole blood and PBMCs to detect Hz-containing leukocytes and CD16 expression on monocytes. C5BL/6 mice were infected with Plasmodium berghei ANKA or P. berghei NK65 and Hz-containing leukocytes were analysed using CD11b and Gr1 expression. Parasitized RBC from infected mice were identified using anti-Ter119 and SYBR green I and were analysed for depolarized Side Scatter. A highly depolarizing RBC population was monitored in an in-vitro culture incubated with chloroquine or quinine.ResultsA flow cytometer can be easily adapted to detect depolarized Side-Scatter and thus, intracellular Hz. The detection and counting of Hz containing leukocytes in fresh human or mouse blood, as well as in leukocytes from in-vitro experiments was rapid and easy. Analysis of CD14/CD16 and CD11b/Gr1 monocyte expression in human or mouse blood, in a mixed populations of Hz-containing and non-containing monocytes, appears to show distinct patterns in both types of cells. Hz-containing pRBC and different maturation stages could be detected in blood from infected mice. The analysis of a highly depolarizing population that contained mature pRBC allowed to assess the effect of chloroquine and quinine after only 2 and 4 hours, respectively.ConclusionsA simple modification of a flow cytometer allows for rapid and reliable detection and quantification of Hz-containing leukocytes and the analysis of differential surface marker expression in the same sample of Hz-containing versus non-Hz-containing leukocytes. Importantly, it distinguishes different maturation stages of parasitized RBC and may be the basis of a rapid no-added-reagent drug sensitivity assay.

Severe malaria increases the list of heme oxygenase-1-protected diseases

Malaria is the most widespread and lethal parasitic disease in the world today, and claims the lives of at least 1 million people every year. The disease is caused by parasitic protozoa of the genus Plasmodium, whose life cycle involves a vertebrate host and a mosquito vector. The parasite undergoes two different stages of infection within the mammalian host. The first stage occurs inside hepatocytes and is clinically asymptomatic. The symptoms associated with the disease appear only during the subsequent stage of infection, which occurs inside erythrocytes. In humans, clinical manifestations of malaria can range from mild to severe malaria, the latter comprising a series of syndromes that include, among others, cerebral malaria (CM) and acute respiratory distress syndrome. Interestingly, although four Plasmodium species infect humans (Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae and Plasmodium ovale), only one of these parasites, P. falciparum, causes fatal disease. The reason(s) for this difference are yet to be fully elucidated. The sequestration of infected erythrocytes in vital organs, a feature unique to P. falciparum infection, has been identifed as a major factor. However, whether this feature causes disease due to mechanical blockage of blood flow or to the release of inflammatory mediators in vulnerable regions caused by local infected-erythrocyte rupture, is still a matter of controversy [1], and increasing evidence is suggesting a role for inflammatory mediators in malaria-associated pathology [2]. Thus, it has been proposed that the pathogenesis of malaria might not differ much from other clinically overlapping systemic diseases caused by other pathogens or even nonpathogen-dependent inflammatory syndromes [3]. Recently, we have used mice infected with Plasmodium berghei ANKA, a rodent model of malaria pathology, to demonstrate that the antiinflammatory molecule heme oxygenase (HO)-1, which degrades heme to generate biliverdin, iron and carbon monoxide (CO), prevents the development of experimental CM (ECM) [4]. Moreover, we demonstrated that exposure to the endproduct of HO-1 activity, CO, also protected mice against ECM [4]. The anti-inflammatory effects of HO-1 activation were first reported in experimental sepsis [5], and later extended to inflammation in general [6]. Again, in these cases, CO was shown to be the mediator molecule between HO-1 activity and protection against inflammation [6]. More recently, it was postulated that the inhibitory effects of CO observed in systemic inflammation, and the role of TNF-induced HO-1, might also play a role in a malarial context [3]. In fact, HO-1 was detected in histological sections from human sepsis and malaria cases [7].