Katrien Deroost

CXCR3 determines strain susceptibility to murine cerebral malaria by mediating T lymphocyte migration toward IFN‐γ‐induced chemokines

Cerebral malaria (CM) results from the binding of infected erythrocytes and leukocytes to brain endothelia. The precise mechanisms underlying lymphocyte recruitment and activation in CM remain unclear. Therefore, the expression of various chemokines was quantified in brains of mice infected with Plasmodium berghei ANKA (PbA). Several chemokines attracting monocytes and activated T‐lymphocytes were expressed at high levels. Their expression was almost completely abrogated in IFN‐γ ligand and receptor KO mice, indicating that IFN‐γ is an essential chemokine inducer in vivo. Surprisingly, the expression levels of chemokines, IFN‐γ and also adhesion molecules in the brain were not lower in CM‐resistant Balb/c and DBA/2 mice compared to CM‐sensitive C57BL/6 and DBA/1 mice, although T lymphocyte sequestration in the brain was significantly less in CM‐resistant than in CM‐sensitive mice. This difference correlated with a higher up‐regulation of the CXC chemokine receptor (CXCR)‐3 on splenic T cells and a higher chemotactic response to IFN‐γ‐inducible protein‐10 (IP‐10) in C57BL/6 compared to Balb/c mice. In conclusion, parasite‐induced IFN‐γ in the brain results in high local expression levels of specific chemokines for monocytes and lymphocytes. The strain‐dependent susceptibility to develop CM is more related to the expression of CXCR3 in circulating leukocytes than to the chemokine expression levels in the brain.

Immunopathology and Dexamethasone Therapy in a New Model for Malaria-associated Acute Respiratory Distress Syndrome

RATIONALE Malaria infection is often complicated by malaria-associated acute respiratory distress syndrome (MA-ARDS), characterized by pulmonary edema and hemorrhages. No efficient treatments are available for MA-ARDS and its pathogenesis remains poorly understood. OBJECTIVES Development of a new animal model for MA-ARDS to explore the pathogenesis and possible treatments. METHODS C57BL/6 mice were infected with Plasmodium berghei NK65, and the development of MA-ARDS was evaluated by the analysis of lung weight, histopathology, and bronchoalveolar lavages. Cytokine and chemokine expression in the lungs was analyzed by reverse transcription-polymerase chain reaction, and the accumulation of leukocyte subclasses was determined by flow cytometric analysis. MEASUREMENTS AND MAIN RESULTS In this model, the pulmonary expression of several cytokines and chemokines was increased to a higher level than in mice infected with Plasmodium chabaudi AS, which does not cause MA-ARDS. By depletion experiments, CD8(+) T lymphocytes were shown to be pathogenic. High doses of dexamethasone blocked MA-ARDS, even when administered after appearance of the complication, and reduced pulmonary leukocyte accumulation and the expression of a monocyte/macrophage-attracting chemokine. CONCLUSIONS We developed a novel model of MA-ARDS with many similarities to human MA-ARDS and without cerebral complications. This contrasts with the more classical model with P. berghei ANKA, characterized by fulminant cerebral malaria. Hence, infection with P. berghei NK65 generates a broader time window to study the pathogenesis and to evaluate candidate treatments. The finding that high doses of dexamethasone cured MA-ARDS suggests that it might be more effective against MA-ARDS than it was in the clinical trials for cerebral malaria.

Hemozoin Induces Lung Inflammation and Correlates with Malaria-Associated Acute Respiratory Distress Syndrome

Malaria-associated acute respiratory distress syndrome (MA-ARDS) is a deadly complication of malaria, and its pathophysiology is insufficiently understood. Both in humans and in murine models, MA-ARDS is characterized by marked pulmonary inflammation. We investigated the role of hemozoin in MA-ARDS in C57Bl/6 mice infected with Plasmodium berghei NK65, P. berghei ANKA, and P. chabaudi AS. By quantifying hemozoin in the lungs and measuring the disease parameters of MA-ARDS, we demonstrated a highly significant correlation between pulmonary hemozoin concentrations, lung weights, and alveolar edema. Histological analysis of the lungs demonstrated that hemozoin is localized in phagocytes and infected erythrocytes, and only occasionally in granulocytes. Species-specific differences in hemozoin production, as measured among individual schizonts, were associated with variations in pulmonary pathogenicity. Furthermore, both pulmonary hemozoin and lung pathology were correlated with the number of infiltrating inflammatory cells, an increased pulmonary expression of cytokines, chemokines, and enzymes, and concentrations of alveolar vascular endothelial growth factor. The causal relationship between hemozoin and inflammation was investigated by injecting P. falciparum-derived hemozoin intravenously into malaria-free mice. Hemozoin potently induced the pulmonary expression of proinflammatory chemokines (interferon-γ inducible protein-10/CXC-chemokine ligand (CXCL)10, monocyte chemotactic protein-1/CC-chemokine ligand 2, and keratinocyte-derived chemokine/CXCL1), cytokines (IL-1β, IL-6, IL-10, TNF, and transforming growth factor-β), and other inflammatory mediators (inducible nitric oxide synthase, heme oxygenase-1, nicotinamide adenine dinucleotide phosphate- oxidase-2, and intercellular adhesion molecule-1). Thus, hemozoin correlates with MA-ARDS and induces pulmonary inflammation.

Replication of Plasmodium in reticulocytes can occur without hemozoin formation, resulting in chloroquine resistance

Lin et al. generate Plasmodium berghei mutants lacking enzymes critical to hemoglobin digestion. A double gene deletion mutant lacking enzymes involved in the initial steps of hemoglobin proteolysis is able to replicate inside reticulocytes of infected mice with limited hemoglobin degradation and no hemozoin formation, and moreover, is resistant to the antimalarial drug chloroquine.

Pathogenesis of malaria-associated acute respiratory distress syndrome

Malaria-associated acute respiratory distress syndrome (MA-ARDS) is an increasingly reported, often lethal, and incompletely understood complication of malaria. We discuss and compare the pathogenesis of MA-ARDS in patients and in different murine models, including recent models without cerebral involvement, and summarize the roles of different leukocyte subclasses, adhesion molecules, cytokines, and chemokines. In patients as well as in mice, severe edema and impaired gas exchange are associated with abundant inflammatory infiltrates consisting of mainly mononuclear cells and parasite sequestration, and the pathogenesis appears different from cerebral malaria (CM). Experimental anti-inflammatory interventions are successful in mice and remain to be validated in patients.

The immunological balance between host and parasite in malaria.

Coevolution of humans and malaria parasites has generated an intricate balance between the immune system of the host and virulence factors of the parasite, equilibrating maximal parasite transmission with limited host damage. Focusing on the blood stage of the disease, we discuss how the balance between anti-parasite immunity versus immunomodulatory and evasion mechanisms of the parasite may result in parasite clearance or chronic infection without major symptoms, whereas imbalances characterized by excessive parasite growth, exaggerated immune reactions or a combination of both cause severe pathology and death, which is detrimental for both parasite and host. A thorough understanding of the immunological balance of malaria and its relation to other physiological balances in the body is of crucial importance for developing effective interventions to reduce malaria-related morbidity and to diminish fatal outcomes due to severe complications. Therefore, we discuss in this review the detailed mechanisms of anti-malarial immunity, parasite virulence factors including immune evasion mechanisms and pathogenesis. Furthermore, we propose a comprehensive classification of malaria complications according to the different types of imbalances.

Improved methods for haemozoin quantification in tissues yield organ-and parasite-specific information in malaria-infected mice

BackgroundDespite intensive research, malaria remains a major health concern for non-immune residents and travelers in malaria-endemic regions. Efficient adjunctive therapies against life-threatening complications such as severe malarial anaemia, encephalopathy, placental malaria or respiratory problems are still lacking. Therefore, new insights into the pathogenesis of severe malaria are imperative. Haemozoin (Hz) or malaria pigment is produced during intra-erythrocytic parasite replication, released in the circulation after schizont rupture and accumulates inside multiple organs. Many in vitro and ex vivo immunomodulating effects are described for Hz but in vivo data are limited. This study aimed to improve methods for Hz quantification in tissues and to investigate the accumulation of Hz in different organs from mice infected with Plasmodium parasites with a varying degree of virulence.MethodsAn improved method for extraction of Hz from tissues was elaborated and coupled to an optimized, quantitative, microtiter plate-based luminescence assay with a high sensitivity. In addition, a technique for measuring Hz by semi-quantitative densitometry, applicable on transmitted light images, was developed. The methods were applied to measure Hz in various organs of C57BL/6 J mice infected with Plasmodium berghei ANKA, P. berghei NK65 or Plasmodium chabaudi AS. The used statistical methods were the Mann–Whitney U test and Pearsons correlation analysis.ResultsMost Hz was detected in livers and spleens, lower levels in lungs and kidneys, whereas sub-nanomolar amounts were observed in brains and hearts from infected mice, irrespectively of the parasite strain used. Furthermore, total Hz contents correlated with peripheral parasitaemia and were significantly higher in mice with a lethal P. berghei ANKA or P. berghei NK65-infection than in mice with a self-resolving P. chabaudi AS-infection, despite similar peripheral parasitaemia levels.ConclusionsThe developed techniques were useful to quantify Hz in different organs with a high reproducibility and sensitivity. An organ-specific Hz deposition pattern was found and was independent of the parasite strain used. Highest Hz levels were identified in mice infected with lethal parasite strains suggesting that Hz accumulation in tissues is associated with malaria-related mortality.

Immunopathological effects of malaria pigment or hemozoin and other crystals

Blood‐stage malaria parasites produce insoluble hemozoin (Hz) crystals that are released in the blood circulation upon schizont rupture. In general, endogenous crystal formation or inhalation of crystalline materials is often associated with pathology. As the immune system responds differently to crystalline particles than to soluble molecules, in this review, the properties, immunological recognition, and pathogenic responses of Hz are discussed, and compared with two other major pathogenic crystals, monosodium urate (MSU) and asbestos. Because of the size and shape of MSU crystals and asbestos fibers, phagolysosomal formation is inefficient and often results in leakage of lysosomal content in the cell cytoplasm and/or in the extracellular environment with subsequent cell damage and cell death. Phagolysosomal formation after Hz ingestion is normal, but Hz remains stored inside these cells for months or even longer without any detectable degradation. Nonetheless, the different types of crystals are recognized by similar immune receptors, involving Toll‐like receptors, the inflammasome, antibodies, and/or complement factors, and through similar signaling cascades, they activate both proinflammatory and anti‐inflammatory immune responses that contribute to inflammation‐associated pathology. BioFactors, 40(1):59–78, 2014

Altered Lipid Composition of Surfactant and Lung Tissue in Murine Experimental Malaria-Associated Acute Respiratory Distress Syndrome

Malaria-associated acute lung injury (MA-ALI) and its more severe form malaria-associated acute respiratory distress syndrome (MA-ARDS) are common, often fatal complications of severe malaria infections. However, little is known about their pathogenesis. In this study, biochemical alterations of the lipid composition of the lungs were investigated as possible contributing factors to the severity of murine MA-ALI/ARDS. C57BL/6J mice were infected with Plasmodium berghei NK65 to induce lethal MA-ARDS, or with Plasmodium chabaudi AS, a parasite strain that does not induce lung pathology. The lipid profile of the lung tissue from mice infected with Plasmodium berghei NK65 developing MA-ALI/ARDS, but not that from mice without lung pathology or controls, was characterized by high levels of phospholipids -mainly phosphatidylcholine- and esterified cholesterol. The high levels of polyunsaturated fatty acids and the linoleic/oleic fatty acid ratio of the latter reflect the fatty acid composition of plasma cholesterol esters. In spite of the increased total polyunsaturated fatty acid pool, which augments the relative oxidability of the lung membranes, and the presence of hemozoin, a known pro-oxidant, no excess oxidative stress was detected in the lungs of Plasmodium berghei NK65 infected mice. The bronchoalveolar lavage (BAL) fluid of Plasmodium berghei NK65 infected mice was characterized by high levels of plasma proteins. The phospholipid profile of BAL large and small aggregate fractions was also different from uninfected controls, with a significant increase in the amounts of sphingomyelin and lysophosphatidylcholine and the decrease in phosphatidylglycerol. Both the increase of proteins and lysophosphatidylcholine are known to decrease the intrinsic surface activity of surfactant. Together, these data indicate that an altered lipid composition of lung tissue and BAL fluid, partially ascribed to oedema and lipoprotein infiltration, is a characteristic feature of murine MA-ALI/ARDS and possibly contribute to lung dysfunction.

Signatures of malaria-associated pathology revealed by high-resolution whole-blood transcriptomics in a rodent model of malaria.

The influence of parasite genetic factors on immune responses and development of severe pathology of malaria is largely unknown. In this study, we performed genome-wide transcriptomic profiling of mouse whole blood during blood-stage infections of two strains of the rodent malaria parasite Plasmodium chabaudi that differ in virulence. We identified several transcriptomic signatures associated with the virulent infection, including signatures for platelet aggregation, stronger and prolonged anemia and lung inflammation. The first two signatures were detected prior to pathology. The anemia signature indicated deregulation of host erythropoiesis, and the lung inflammation signature was linked to increased neutrophil infiltration, more cell death and greater parasite sequestration in the lungs. This comparative whole-blood transcriptomics profiling of virulent and avirulent malaria shows the validity of this approach to inform severity of the infection and provide insight into pathogenic mechanisms.