The first observations of black pigment in necroptic spleens and brains go back to the 18th Century (reviewed in ). About 130 years later, a publication mentioned brown-grey colourations of brain, spleen and liver, which turned out to arise from pigment deposition. At first believed to be melatonin, it was later linked to a parasitic disease. Presently, many in vitro and ex vivo immunomodulating effects have been ascribed to Hz [4–7]. However, data about the fate and properties of Hz in the in vivo situation are still scarce. Hz is released in the circulation in considerable amounts after schizont rupture where it may interact with a whole range of different cell types. The majority of the liberated Hz is presumably captured and phagocytosed by circulating and tissue resident monocytes/macrophages in which it can persist for a long time. In this way, Hz may be capable of causing considerable inflammation that might progress to tissue injury. In this study, techniques for sensitively quantifying the amount of Hz in tissues were examined and the organ-specific Hz content was compared between parasite species with a varying degree of pathogenicity.
As Hz crystals were observed on unstained cryosections from livers, spleens, lungs and kidneys, a technique for estimating the amount of Hz in these sections by densitometric analysis was developed. As Hz is proportionally distributed throughout the liver, the estimation of the amount of Hz by densitometry was quite reliable. In other organs, however, Hz was found in specific structures such as the red pulp in the spleen, the interstitial tissue in the lungs or presumably the glomeruli in the kidneys. This may in part be attributed to the differential localization of tissue-resident phagocytes. This implies that Hz distribution is a confounding factor for the accuracy of the Hz measurements on organ cryosections by densitometry. In addition, this technique is time-consuming, labour-intensive, semi-quantitative and not suitable for organs with a low Hz content and was thus not further explored. Therefore, a more sensitive, analytical and quantitative method for determining the Hz content in tissues was investigated. To isolate Hz from organs, a protocol described by Sullivan and colleagues  was modified. The main adaptation was the digestion of the homogenates with proteinase K. This digestion eliminated high background signals, which were presumably due to the binding of Hb to otherwise insoluble extracellular matrix components. Upon conversion of the isolated Hz into soluble haematin, a chemo-luminescence assay was used for quantification. This assay was based on the method of Schwarzer et al. and adapted to microtiter plate format. The obtained sensitivity with the optimized protocol was lower compared with the haem-enhanced luminescence assay described by Schwarzer et al. This was not due to quenching of the luminescence signal by SDS nor was it caused by the altered time frame during which the emitted light was measured (two seconds/sample versus approximately ninety six seconds/plate), but probably originated from the use of different luminescence detector systems (cuvette system versus microplate reader). Nevertheless, the microplate-adjusted approach offers the advantage of measuring several samples in varying concentrations simultaneously with a sensitivity that is optimal for the quantification of Hz in malaria-infected organs.
As an application, the distribution of Hz throughout the body of infected mice was studied and compared between diverse parasite strains with varying pathogenicity. Sullivan and colleagues already quantified the Hz content in brains, livers and spleens of mice [15, 29] and in human placentas , but no detailed comparison between organs and between parasite species was described. Almost 95% of the total pool of Hz was found in livers and spleens. This was expected as large volumes of blood are filtered through these organs and both contain a vast population of tissue-resident monocytes/macrophages capable of rapidly removing the crystalline material from the circulation by means of phagocytosis. It was also important to consider the liver and spleen sizes when determining the total Hz amounts, as these sizes evolve in a different way during infection with different parasites (i.e. induction of hepatosplenomegaly by PcAS). As the absolute Hz concentration in the organs could be determined by the luminescence assay, this was easily taken into account by multiplication with the organ weights.
Furthermore, substantial amounts of Hz were detected in lungs of malaria-infected mice. In a new mouse model of MA-ARDS , considerable amounts of Hz were observed on histological sections of the lungs. By quantifying the Hz content in the lungs, significantly higher Hz levels were validated in lungs from P. berghei-infected mice (lung pathology) compared to PcAS-infected mice (no lung pathology), indicating that Hz may have a role in the development of malaria-associated lung disease.
Low but detectable amounts of Hz were found in kidneys, hearts and brains of malaria-infected mice. Most Hz was detected in kidneys and hearts from PbNK65-infected mice ten days post-infection compared with PbANKA and PcAS-infected mice seven to eight and ten days post-infection, respectively. However, a different pattern was observed in the brains, i.e. Hz was undetectable in brains from PcAS-infected mice whereas similar amounts of Hz were detected in brains of PbNK65 and PbANKA-infected mice. A possible explanation for this difference is their diverse parasite synchronicity. At the moment of sacrificing the mice and organ removal, the PcAS-parasites in the circulation were all in the ring and young trophozoite stage. As these developmental stages do not yet contain abundant Hz [3, 10], it seemed reasonable that Hz was not detected in brains from mice infected with this parasite species. It is also possible that no Hz was detected because PcAS-parasites may not sequester in the brains as is the case for Plasmodium vivax-infected erythrocytes . On the contrary, several developmental stages of P. berghei parasites are found in the circulation simultaneously and accumulation of P. berghei in the brain is still a debated issue. The observation of similar brain Hz contents in PbANKA and PbNK65-infected mice cannot be explained by their parasitaemia levels as significantly higher parasitaemias were found in mice that were infected with PbNK65 than in mice infected with PbANKA. The data however do suggest that Hz as such is not sufficient for the development of this immunopathology as PbNK65-infected C57BL/6 J mice do not develop cerebral complications . These data are in contrast with data from Coban et al. and Sullivan et al. who found that brains from mice with cerebral pathology contained more Hz than healthy brains from infected mice. However, this may be explained by differences in the timing of analysis after infection and in the mouse or parasite strains used in the studies.
Organ-trapped Hz may originate from two sources. As free Hz is rapidly removed from the circulation, it is found either inside phagocytes or inside cyto-adhering iRBCs along the endothelial lining of the organs’ microvasculature. Systemic perfusion removes circulating iRBCs but not sequestering iRBCs or Hz inside resident phagocytes, although inadequate perfusion can result from obstruction due to organ-specific cyto-adherence and haemorrhages. Furthermore, it is still not completely clarified if sequestration by murine malaria parasites occurs and which organs are the main targets. Local parasite accumulation has been demonstrated in brains and lungs of PbANKA-infected mice suffering from cerebral symptoms [18, 30], but no reports exist on PbNK65-parasite sequestration.
After calculating the total amount of Hz in the mice, it was found that PbANKA and PbNK65-infected mice contained similar amounts of Hz at comparable parasitaemia levels. This suggests that both parasites produced similar amounts of Hz, or that their schizonts presumably consumed comparable amounts of Hb. On the contrary, lower amounts of Hz were retrieved in PcAS-infected mice despite of similar peripheral parasitaemia. Several explanations can be given for this finding. PcAS-parasites may produce less Hz, e.g. by digesting less Hb or by using other haem detoxification mechanisms (transport of haem out of the food vacuole or anti-oxidative defense mechanisms of the parasite) or PcAS Hz could be more easily degraded. Interestingly, Noland et al. demonstrated that Hz crystals from different Plasmodium species have different shapes and dimensions, supporting the notion that Hz from different species may have different properties. In addition, Hz contents are variable in RBC infected with different Plasmodium falciparum strains .
Another possibility is that peripheral parasitaemia, estimated by counting the percentage of iRBCs by microscopic analysis of Giemsa-stained blood smears, are not a true reflection of the total parasite biomass as they do not take sequestered parasites into account. Consequently, it is possible that PcAS-infected mice contain less Hz because of lower total parasite burdens. These observations may well translate to the situation in human malaria, where various parasite species have different degrees of virulence. Total parasite biomass in P. falciparum infections is higher than peripheral parasitaemia levels and the difference between these two parameters increases with disease severity . Similarly, Hz-containing peripheral leukocytes are a marker for disease severity [34–36], and accumulation of Hz in brain micro-vessels is associated with a subtype of cerebral malaria . No data are available yet about total parasite burdens in P. vivax-infections and it is still questionable if P. vivax-iRBCs can adhere to the endothelial micro-vascular lining. However, cytoadhesion of P. vivax-infected erythrocytes was demonstrated in vitro and, despite of the absence of sequestration in the brain , it was hypothesized that parasitized RBCs might sequester in lungs from patients with P. vivax malaria . Similarly, very little knowledge exists on the role of Hz in P. vivax infections.
Besides differences in pathogenicity, another interesting difference between P. berghei and PcAS is that PcAS can be cleared from the circulation in several mouse strains, including C57BL/6 mice, whereas PbANKA and PbNK65 cannot. The amounts of Hz produced by these parasites may also contribute to these differences, as Hz is known to suppress macrophage activity in vitro and in vivo. Interestingly, Spaccapelo et al. found that plasmepsin 4-deficient PbANKA-parasites, which produce less Hz, cause less immunopathology and are more easily cleared by some mouse strains .