Background
With growing world populations in tropical countries, every year more people become exposed to malaria. As many areas of endemic malaria transmission overlap with regions of poverty, direct and indirect burdens of this infectious disease are important. In particular, a small percentage of the malaria-infected patients develop life-threatening complications such as severe malarial anaemia, encephalopathy, placental malaria or respiratory problems, even if they have similar peripheral parasitaemia levels compared to patients with mild or asymptomatic malaria [
1]. Efficient adjunct therapies against these immunopathological complications are still not available. Therefore, studying the mechanisms of disease development in severe malaria is paramount.
Inside the red blood cell (RBC), almost 80% of the haemoglobin (Hb) is degraded, which means that high amounts of toxic haem (which is rapidly oxidized to haemin) are liberated in the food vacuole of the parasite capable of generating reactive oxygen species and damaging cell membranes and proteins [
2,
3]. As a detoxification mechanism, the parasite biocrystallizes the haemin molecules into insoluble haemozoin (Hz) crystals or malaria pigment [
4,
5]. When the schizont ruptures, Hz is released into the circulation and is rapidly removed by phagocytes inside several organs. Multiple
in vitro and
ex vivo pro-inflammatory and immunosuppressive effects of Hz have been described (reviewed in [
4‐
7]). However, few
in vivo data about the effects of Hz on the immune system exist. As large amounts of Hz are produced during infection and accumulate inside multiple organs, Hz may be important for the progress towards malaria-associated pathologies. This hypothesis is further strengthened by the fact that abundant Hz has been observed in brains [
8‐
10] and placentas [
11‐
13] from malaria patients with cerebral and placental complications, respectively. In addition, Hz was detected in brains of mice with cerebral symptoms [
14,
15] and in lungs of mice with malaria-associated acute respiratory distress syndrome (MA-ARDS) [
16].
Experimental mouse models offer useful tools to study malaria-related disease mechanisms. Depending on the mouse-parasite combination, different aspects of human malaria can be mimicked and investigated, even if these models are not exact replicas and should thus be extrapolated with caution. In this study, C57BL/6 J mice were infected with three different parasite strains with a varying degree of pathogenicity. The
Plasmodium berghei ANKA
(Pb ANKA
) parasite induces typical symptoms of cerebral malaria (CM), such as paralysis or coma and mice succumb within seven to nine days. In this mouse model, the pathology critically depends on activation of leukocytes, including CD8
+ T cells, and a local inflammatory reaction [
1]. Although sequestration of
Plasmodium falciparum in the brain is strongly associated with CM in patients, it is unclear whether specific cyto-adherence of
Pb ANKA occurs in the brain [
17]. However, local parasite accumulation in the brain is thought to be an important feature of this model [
18]. Mice infected with
Plasmodium berghei NK65
(Pb NK65
) do not develop such an encephalopathy but rather die from severe respiratory problems between nine and eleven days post-infection [
16]. This respiratory pathology closely resembles human MA-ARDS, as in both mice and patients leukocytes (predominantly macrophages and lymphocytes) and infected RBCs (iRBCs) accumulate in the lungs, resulting in the disruption of endothelial barriers, severe edema and intra-alveolar hyaline membrane formation [
16,
19,
20].
Plasmodium chabaudi AS (
Pc AS)-infections are self-limiting and the mice are able to recover. Moreover, C57BL/6 mice mount a protective immune response against
Pc AS-parasites mediated by phagocytes, CD4
+ T cells and specific antibodies, which is very similar to the immune response generated against
P. falciparum in humans [
21].
To investigate the organ-specific Hz deposition in these three mouse models, novel techniques are described in the present study to accurately quantify the Hz content in tissues. These methods were implemented to compare the amount of Hz between various organs and between similar organs from mice infected with parasites of different pathogenicity. Most Hz was found in livers and spleens. Far less Hz was detected in lungs and kidneys, whereas limited amounts of Hz were observed in hearts and brains, irrespectively of the parasite species. In addition, more Hz was found in mice infected with Pb NK65 or Pb ANKA compared with Pc AS-infected mice despite of similar peripheral parasitaemia levels.
Methods
Chemical products
All chemicals were purchased from Sigma-Aldrich (Bornem, Belgium), unless otherwise stated.
Mice and parasites
C57BL/6 J mice (seven to nine weeks old) were obtained from Janvier (Le Genest-Saint-Isle, France) and placed in a conventional animal house with food and water ad libitum. Parasite growth in mice was supported by supplementing the drinking water with 0.375 mg/mL 4-amino benzoic acid. Mice were intraperitoneally infected with 104 iRBCs by serial passage of tail vein blood obtained from a mouse that had been infected with one of the following parasite strains: Pc AS, Pb NK65 (kind gifts of the late Prof. D Walliker, University of Edinburgh, Scotland, UK) or Pb ANKA (Cl15CY1, a kind gift of Prof. C Jansse, Leiden University Medical Centre, The Netherlands). The percentage of infected erythrocytes in the peripheral blood was determined by microscopic analysis after Giemsa staining. Mice were sacrificed at the indicated time points after infection and blood was removed by heart puncture. Mice were perfused with Dulbecco’s phosphate-buffered saline (PBS) (Lonza, Verviers, Belgium) to remove circulating iRBCs from the organs. Livers, spleens, kidneys, lungs, hearts and brains were removed, weighed and stored at -80°C until further analysis. A part of the liver, spleen, lung and kidney was embedded in Tissue-Tek O.C.T. Compound (Sakura Finetek Europe B.V., Alphen aan den Rijn, The Netherlands) and frozen in liquid nitrogen-cooled isopentane for histological analysis. All experiments were approved by the local ethical committee (License LA121251, Belgium).
Haemozoin quantification in organ cryosections by densitometric analysis
Cryosections with a thickness of 7 μm were prepared from frozen livers, spleens, lungs and kidneys and imaged by light microscopy. Transmitted light images were taken through a 20x/0.8 Plan-Apochromat objective of an Axiovert 200 M microscope equipped with an AxioCam MRm camera (Zeiss, Göttingen, Germany). For Hz quantification on liver sections, images were obtained from two rows of three consecutive fields. The densitometric analysis was performed with the AxioVision 4.6 software with a home-written script and the relative quantity of Hz/μm
2 was calculated with the formula as shown in Figure
1G. The densitometric value (DV) of a pixel reflects the intensity of transmitted light at this position in the section. The densitometric background (DB) was determined in each picture by calculating the mean DV of all pixels with a DV above an empirically determined threshold. To test the linearity of the densitometric method to measure Hz on cryosections, gelatin blocks containing different concentrations of synthetic Hz (sHz) were analysed. sHz was prepared as described previously [
22] and was homogenized and added in different concentrations to a 10% gelatin-PBS solution at 37°C in a 24-well plate. Upon solidification on ice (to prevent sedimentation of sHz), sHz-containing gelatin blocks were cut out of the wells, embedded in O.C.T and frozen at -80°C. For each concentration, five to six images were analysed from one section/sHz block and this was done for two blocks.
Haem quantification by colorimetric analysis and haem-enhanced luminescence
The Fe-ions in the haemin molecules that constitute the Hz crystal are in the oxidized state (Fe
3+). Therefore, a dilution series of a haematin stock solution (10 μM – 1.2 nM), prepared by dissolving haemin in 100 mM NaOH, 2% SDS and 3 mM EDTA, was used as a standard to compare methods. In an alkaline environment, haematin produces a brown colour measurable spectrophotometrically (Biotech Powerwave XS) at 405 nm [
23]. Background absorbance was evaluated from a blank sample and subtracted from the measurements. The method for quantification by luminescence was based on the method of Schwarzer
et al.[
24] and was optimized for working in a 96-well plate and modified according to Yuan
et al.[
25]. Different concentrations of haematin were added in 96-well plates suitable for luminescence (Perkin Elmer, Waltham, MA, USA) and diluted in a solution containing NaOH and Na
2CO
3 (four volumes of 100 mM NaOH, 2% SDS and 3 mM EDTA and one volume of 1 M Na
2CO
3, pH 10.4) (final volume 50 μL). After addition of 100 μL luminol (100 μg/mL 3-aminophtalhydrazide) and 100 μL of peroxide (7%
tert-butyl hydroperoxide), both dissolved in the NaOH/Na
2CO
3-solution, light emitted in the presence of Fe
3+ (present in the haematin core) was measured during one second using a Thermo Luminoskan Ascent apparatus. Peroxide catalysis into oxygen by Fe
3+ is a fast reaction. Therefore, special care was taken to keep the time between the addition of the peroxide and the luminescence measurements minimal and as similar as possible between the different wells (maximum time deviation between individual wells was eight seconds). A sigmoidal relationship between the haematin concentration and the luminescence (events/sec) was obtained. Background luminescence was evaluated from a blank sample and subtracted from the measurements. The above method for haem-enhanced luminescence was used for all measurements unless differently stated. The time-dependence of the luminescence signal was measured with 125 nM haematin in duplicate every ten seconds for eight minutes during a kinetic reading without any other samples in the plate to allow fast repetitive reading of these two wells. A lag-time of twelve seconds existed between the addition of the peroxide and the start of the measurement, which was partly attributable to a shaking step.
Haemozoin determination in tissues and trophozoites
To extract haemozoin from perfused mouse tissues, approximately 30 – 60 mg (liver, spleen, kidney or lung), half brain or a full heart were homogenized with the Precellys Lysing Kit (VWR, Leuven, Belgium) in minimum five volumes of a solution containing 50 mM Tris/HCl pH 8.0, 5 mM CaCl2, 50 mM NaCl and 1% Triton X-100. The homogenate was supplemented with 1% Proteinase K and incubated overnight at 37°C. The next day the proteinase K digest was sonicated (VialTweeter, Hielscher Ultrasonics GmbH, Teltow, Germany) for 1 min (10 W, pulse 0.5 sec) and centrifuged at 11,000 x g for 45 min. The supernatant was discarded and the pellet was washed three times in 100 mM NaHCO3, pH 9.0 and 2% SDS with subsequent sonication and centrifugation for 30 min to remove degraded tissue, free haem and Hb. After the third wash, the pellet (Hz) was dissolved and sonicated in 100 mM NaOH, 2% SDS and 3 mM EDTA to form haematin and centrifuged to pellet any remaining insoluble material. To confirm that the isolated material was indeed Hz, it was examined for its birefringence character. For this purpose, isolated Hz that was washed three times as described above, was subsequently washed in distilled H2O to remove the salts, smeared on a glass slide and monitored by polarized light microscopy with a 40x/1.3 oil EC Plan-Neofluar objective of an Axiovert 200 M microscope.
To isolate Hz from trophozoites, heparinized blood was obtained by heart puncture from
Pb NK65-infected mice and trophozoites were cultivated
ex vivo overnight and harvested as described [
26]. After determination of the total red cell number and the percentage of iRBCs, Hz was extracted from the cells as described above but without proteinase K treatment and subsequently dissolved as described.
The extracted Hz was measured in different dilutions with the above-mentioned protocol for haem quantification by luminescence. A dilution series of haematin (10 μM – 1.2 nM) was used as a standard. The unknown Hz concentration was calculated from the calibration curve of the haematin concentration (nM) versus luminescence (events/sec). Background luminescence was evaluated from a blank sample and subtracted from the measurements. The amount of Hz (fmol or pmol haematin/mg tissue) was multiplied with the total weight of the concerning organ and expressed as pmol or nmol haematin/organ. An accuracy limit was estimated for each organ separately.
Statistical analysis
P-values for the differences between two groups were calculated with the Mann–Whitney
U-test, using the GraphPad Prism software (GraphPad Software, San Diego, CA, USA). The same software was used for linear regression analyses and for calculating Spearman correlation coefficients. The slopes of the individual regression lines of the different groups were compared online [
27]. A
p-value less than 0.05 (
p < 0.05) was taken as statistically significant.
Discussion
The first observations of black pigment in necroptic spleens and brains go back to the 18
th Century (reviewed in [
5]). 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 [
15] 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.[
24] 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 [
11], 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
Pc AS). 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 [
16], 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
Pc AS-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
Pb NK65-infected mice ten days post-infection compared with
Pb ANKA and
Pc AS-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
Pc AS-infected mice whereas similar amounts of Hz were detected in brains of
Pb NK65 and
Pb ANKA-infected mice. A possible explanation for this difference is their diverse parasite synchronicity. At the moment of sacrificing the mice and organ removal, the
Pc AS-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
Pc AS-parasites may not sequester in the brains as is the case for
Plasmodium vivax-infected erythrocytes [
10]. 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
Pb ANKA and
Pb NK65-infected mice cannot be explained by their parasitaemia levels as significantly higher parasitaemias were found in mice that were infected with
Pb NK65 than in mice infected with
Pb ANKA. The data however do suggest that Hz as such is not sufficient for the development of this immunopathology as
Pb NK65-infected C57BL/6 J mice do not develop cerebral complications [
16]. These data are in contrast with data from Coban
et al.[
14] and Sullivan
et al.[
15] 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
Pb ANKA-infected mice suffering from cerebral symptoms [
18,
30], but no reports exist on
Pb NK65-parasite sequestration.
After calculating the total amount of Hz in the mice, it was found that
Pb ANKA and
Pb NK65-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
Pc AS-infected mice despite of similar peripheral parasitaemia. Several explanations can be given for this finding.
Pc AS-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
Pc AS Hz could be more easily degraded. Interestingly, Noland
et al.[
31] 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 [
32].
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
Pc AS-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 [
33]. 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 [
37]. 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[
38] and, despite of the absence of sequestration in the brain [
10], it was hypothesized that parasitized RBCs might sequester in lungs from patients with
P. vivax malaria [
39]. 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
Pc AS is that
Pc AS can be cleared from the circulation in several mouse strains, including C57BL/6 mice, whereas
Pb ANKA and
Pb NK65 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[
40] and
in vivo[
41]. Interestingly, Spaccapelo
et al. found that plasmepsin 4-deficient
Pb ANKA-parasites, which produce less Hz, cause less immunopathology and are more easily cleared by some mouse strains [
42].
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
KD participated in the design of the study, performed the animal experiments, optimized the luminescence technique, analysed and interpreted the data, performed the statistical analysis and drafted the manuscript. NL participated in Hz analysis. SN operated the microscope and designed the script for the densitometric analysis of Hz on pictures from organ cryosections. EM participated in optimizing the luminescence technique. GO participated in study design, manuscript writing and provided critical support. PVDS conceived and participated in the design of the study, optimized the method for densitometric analysis of Hz in cryosections and participated in interpreting the data and drafting the manuscript. All authors read and approved the final manuscript.