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Role of hemozoin in malaria immunopathologies

Book - Dissertation

Malaria is a life-threatening disease caused by Plasmodium parasites and transmitted by Anopheles mosquitoes. According to the annual world malaria report of the World Health Organization, an estimated 216 million people were clinically affected by malaria in 2010 and approximately 655 000 of these patients died from severe complications. Intraerythrocytic parasite replication, cytoadherence to the microvascular endothelium of several organs to evade antiparasite immunity and subsequent rupture of mature schizonts are responsible for the clinical symptoms observed during malaria. These symptoms may vary from fever and mild disease to life-threatening complications such as severe anemia, cerebral malaria and malaria-associated acute respiratory distress syndrome (MA-ARDS). Considerable amounts of hemozoin (Hz) crystals are produced to detoxify hemin during hemoglobin digestion, are released in the circulation and are rapidly cleared by phagocytosis. In the phagosome, Hz cannot be degraded and remains stored long after parasite clearance. Due to the dual role of Hz on the immune system, Hz may modulate the immunological balance and determine the outcome of infections. Therefore, we decided to investigate the role of Hz in malarial immunopathologies in more detail in this doctoral thesis.For a better understanding of the immunological balance between host and parasite, a literature survey was conducted. The results of more than 500 PubMed studies with clinical data or mouse malaria models were combined into a database containing 2451 entries. This database enabled us to generate an extensive overview about the different aspects of antimalarial immunity, immune evasion and immunopathology, and to yield insights into the mechanisms that determine the outcome of malaria infections. Based on these insights, we classified the different malaria complications according to the involved immunological balances.In the first experimental part of this doctoral thesis, we developed and optimized reliable techniques to measure Hz in tissues. Since Hz was visible on unstained organ cryosections, we developed a densitometric technique to estimate Hz on transmitted light images of organ sections. As this method was only semi-quantitative, influenced by the Hz distribution pattern, and had a low sensitivity, a heme-enhanced chemoluminescence assay was optimized for use in a 96-well plate and towards higher sensitivity. The latter technique was applied to quantify Hz in different organs of malaria-infected mice. More than 90% of Hz was detected in liver and spleen, limited amounts in lungs and kidneys, whereas only low amounts were found in heart and brain. This distribution was similar for mice infected with parasite species with a varying degree of pathogenicity. After calculating the total Hz content, we found significantly higher total Hz levels in mice infected with lethal P. berghei compared to mice infected with non-lethal and self-resolving P. chabaudi AS (PcAS). These data show that Hz accumulates in different organs, that the majorityis stored in liver and spleen, two organs with a high phagocytic capacity, and that high organ-specific Hz loads may lead to more severe disease.In a second part of this thesis, we investigated the role of Hz in malaria complications. Previously, a new animal model was developed by our group that strikingly resembles the histopathology of MA-ARDS as observed in patients. Lungs from C57BL/6 mice infected with P. berghei NK65 (PbNK65) had increased weights, hemorrhages and pathological edema. We found significantly higher amounts of Hz in lungs from mice developing MA-ARDS compared to mice without pulmonary complications. Histological analysis indicated that Hz was localized inside infected erythrocytes and monocytes/macrophages. The quantitative differences were mainly due to higher Hz production by P. berghei-parasites and higher pulmonary parasite burdens. Furthermore, Hz levels, edema and inflammation in the lungs were tightly associated with each other, and the causative role of Hz in pulmonary inflammation was confirmed by intravenous injection of P. falciparum-derived Hz. These data indicate that Hz-induced pulmonary inflammation is associated with MA-ARDS.In a last part of this dissertation, the role of Hz was investigated in malaria-associated liver injury. Elevated liver enzymes were detected in serum of mice infected with P. berghei ANKA (PbANKA), PbNK65 and PcAS, and were significantly higher in PcAS-infected mice despite of their lower hepatic Hz load. In PcAS-infected mice, a strong correlation was found between hepatic Hz concentrations, hepatocyte damage and inflammation, and the role of Hz on inflammatory gene transcription in the liver was illustrated by intravenous injection of P. falciparum-derived Hz. In contrast, no correlations were found between Hz, pathology and inflammation with P. berghei. However, a negative correlation was found between Hz and major histocompatibility complex (MHC) class II expression by monocytes/macrophages, indicating that high amounts of Hz, as observed in the livers of P. berghei-infected mice, may downregulate pro-inflammatory immune mechanisms. As a conclusion, Hz is an important inflammatory virulence factor in MA-ARDS and hepatocellular dysfunction and Hz may differentially influence immunopathology in a concentration-dependent manner.
Publication year:2012