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Interplay of hemozoin and host factors in malaria immunopathogenesis

Book - Dissertation

Malaria is a widespread mosquito-borne infectious disease affecting about 500 million people yearly and resulting in over 1 million of deaths, particularly in sub-Saharan Africa. The majority of malaria-related mortalities is caused by infections of Plasmodium falciparum and occurs mostly in young infants under the age of five years. Also, primigravidae are at increased risk of pregnancy-associated malaria. Although people living in endemic regions do acquire clinical immunity when frequently infected, no effective malaria vaccine is yet available. An inadequate understanding of the immune mechanisms leading to the development of natural immunity is a critical factor contributing to this failure. To date, RTS,S is the most clinically advanced subunit candidate-vaccine in the development pipeline and provides ± 50% protection.Upon the bite of an infected mosquito,sporozoites enter the bloodstream and migrate to the liver. After passing through a single phase of asymptomatic schizogony in the hepatocytes, merozoites are released in the blood circulation. This asexual blood-stage parasite infects the red blood cell (RBC) and starts a cycle of erythrocytic schizogony, accompanied by the induction of malaria disease symptoms ranging from fever to life-threatening pathology, e.g. severe malaria anemia and cerebral malaria. Many of the severe malaria-associated pathologies have an immunological basis, as an inappropriately regulated proinflammatory immune response elicited by parasitized RBCs and high doses of parasite toxins significantly contributes to pathogenesis. Matrix metalloproteinases (MMPs) are host endopeptidases capable of degrading a broad range of substrates, e.g. extracellular matrix proteins but also bioactive molecules. After activation by unsealing the catalytic site from the propeptide, the enzymes intervene in many physiological processes, e.g. morphogenesis, inflammation and wound repair. Their activity is fine-tuned by endogenous inhibitors, e.g. the tissue inhibitors of metalloproteinases (TIMPs). However, inappropriately regulated MMP activity is implicated in a variety of pathological conditions, such as uncontrolled inflammation, cancer, vascular and neurodegenerative disorders and autoimmune diseases. MMPs may also contribute to infection-related pathology, e.g. by favoring pathogen dissemination throughout the body and by opening barriers to immunoprivileged sites. In malaria, trypanosomiasis, leishmaniasis and toxoplasmosis, a common denominator is meningoencephalitis, characterized by the sequestration and/or migration of leukocytes and/or parasites across the blood-brain barrier (BBB), probably assisted by the proteolytic activity of MMPs. During its development inside the RBC, the parasite digests in its parasitophorous vacuole up to 80% of the host cell hemoglobin as a major nutrient source. During this catabolic process, toxic free heme is liberated and rapidly detoxified into a brownish insoluble crystal called hemozoin. Upon rupture of the schizont, hemozoin is released into the circulation and this malaria pigment catalyzes the production of reactive oxygen species and triggers immunomodulation linked to severe malaria pathology. In malaria, hemozoin is reported as an MMP-9 agonist. Based on the association of activated MMP-9 with several hemolytic diseases, e.g. experimental cerebral malaria, together with the resemblance of the hemopexin-domain of MMP-9 and the plasma hemopexin protein, the physiological scavenger of heme, it was intriguing to further decipher the link between hemozoin and MMP-9. Furthermore, hemozoin accumulation is observed in brain vessels of human malaria patients. By performing in vitro incubation experiments, it was shown that a direct interaction between ß-hematin or synthetic hemozoin, which is structurally identical to natural hemozoin, and the MMP-9 hemopexin domain provokes autocatalytic aminoterminal processing of the propeptide of MMP-9. The truncated enzyme was still catalytically inactive and protein sequencing by Edman degradation revealed that the ß-hematin-induced cleavage site was identical to the first cleavage site of MMP-3, a well-known activator of MMP-9 which cleaves the prodomain in two steps. This ß-hematin-mediated allosteric priming of proMMP-9 significantly enhanced the kinetics of its activation by the catalytic domain of MMP-3. The use of physiological concentrations of ß-hematin might imply some biological relevance for this process occurring in vivo, e.g. in cerebral malaria where disruption of the BBB is central to the pathogenesis. Although hemozoin was initially described as a metabolically inert waste product of the parasite, there is compelling evidence that this crystalloid pigment is involved in malaria-associated immunopathology. Hemozoin was observed to accumulate in bone marrow and to substantially contribute to severe malaria anemia that is caused by both loss and inadequate production of RBCs. In view of the hemozoin-induced priming of MMP-9 activation, the question was raised whether active MMP-9 might cleave and inactivate major erythropoietic growth factors, e.g. erythropoietin (Epo) and Growth Arrest Specific 6 (Gas6). No processing of these factors, however, was observed after overnight incubation with active MMP-9. On top of its function as a major MMP-9 endogenous inhibitor, TIMP-1 was documented as having erythroid-potentiating activity. By studying an experimental Plasmodium chabaudi (PcAS) infection in TIMP-1-/- mice, it became evident that deletion of TIMP-1 had no aggravating effect on the course of anemia. Taken together, these data suggest that both MMP-9 and TIMP-1 have no profound role in the regulation of erythropoietic processes. The molecular mechanisms through which hemozoin mediates its immunological effects are still not elucidated. This parasite-host interplay was suggested to involve Toll-like receptor 9 (TLR9), although it is still not clear whether hemozoin itself is sensed byTLR9, or if this crystalloid pigment functions as a vector that targets parasite DNA to TLR9. In addition, controversies exist on the significance of this pathogen recognition receptor in malaria immunological and pathogenic processes. To evaluate the in vivo role of TLR9 during malaria infection, a non-lethal PcAS infection was studied in genetically modified mice that lack TLR9. In comparison with commercial C57Bl/6 wild type (WT) control mice, these TLR9-/- mice initially showed an increased susceptibility phenotype, characterized by elevated acute and chronic parasitemia levels as well as a Th2-skewed antibody profile. However, these differences disappeared when WT and TLR9-/- mice derived from one intercross generation between both strains were compared. Although the TLR9-/- mice were originally received as being backcrossed onto C57Bl/6 for at least 10 generations, reassessment of the genetic background via single nucleotide polymorphism (SNP) analysis revealed that these TLR9-/- mice were definitely not congenic to C57Bl/6. Indeed, these knockout (KO) mice still carried large regions of 129 DNA, probably originating from the 129 stem cell donor in which the gene is mutated. The degree of C57Bl/6 DNA in the TLR9-/- mice was calculated to be 69%, indicating a maximum of two backcross generations to the C57Bl/6 inbred strain had occurred. Hence, the observed susceptibility phenotype against PcAS in the TLR9-/- mice was not induced by the null mutation, but was probably provoked by the heterogeneous genetic background since the 129 strain is reported to be susceptible for PcAS infection. The random shuffling of the 129 genes during meiosis might have caused the loss of phenotypic differences between the additionally backcrossed TLR9-/- mice and their matched WT controls. These findings illustrate the importance of studying gene function in congenic strains, i.e. strains that are, as a result of repeatedly backcrossing mice heterozygous for a mutation to an inbred stain, genetically identical to this inbred strain except for the targeted gene and its flanking region derived from the donor strain. This strategy will minimize phenotypic line differences due to background genes and thereby reduce the chance of misinterpretations. Furthermore, since most genes do not function in isolation but instead have to be considered in a multigenetic context, it is appropriate to analyze the effects of null mutations on different congenic genetic backgrounds. Solutions for the problem of the flanking genes are the conditional KO models as well as the use of co-isogenic lines (ES cells from donor strain with desired genetic background). Unfortunately, it has to be acknowledged that in many research papers on KO studies, only a sloppy description of the used mice is provided, which complicates rederivation of the identity of the mice and confounds reproducibility. This might explain the discrepancies, for example, on the role of TLR9 during a malaria infection. Hence, researchers need to be aware of the profound effects of the genetic background on gene function and we advocate that for all experiments designed with genetically modified mice, a detailed description on the origin and genetic background of both the control WT and the mutant strain is presented. In conclusion, MMPs may contribute to infection-related pathology due to their elevated and unbalanced proteolytic activity. For instance, we found that hemozoin is able to prime the activation of MMP-9, one of the main inflammatory MMPs. However, further elucidation of the regulation and functional roles played by MMPs/TIMPs during malaria may improve insights into immunopathogenic processes. Studies with MMP KO mice can help unravel gene function, yet it is important to evaluate KO phenotypes on a well-defined genetic background identical to the control mice. Indeed, the heterogeneity in the genetic background might be a confounding factor, for example, to explain the current controversies on the role of TLR9 in malaria infections. Hence, it is of critical importance to describe the used mice in full detail when publishing data of studies with genetically modified animals.
Publication year:2011