Statement of equal author contribution: T.B and D.C. contributed equally to this study.
Infection with the malaria parasite, Plasmodium, is associated with a strong inflammatory response and parasite cytoadhesion (sequestration) in several organs. Here, we have carried out a systematic study of sequestration and histopathology during infection of C57Bl/6 mice with Plasmodium chabaudi AS and determined the influence of the immune response. This parasite sequesters predominantly in liver and lung, but not in the brain, kidney or gut. Histopathological changes occur in multiple organs during the acute infection, but are not restricted to the organs where sequestration takes place. Adaptive immunity, and signalling through the IFNγ receptor increased sequestration and histopathology in the liver, but not in the lung, suggesting that there are differences in the adhesion molecules and/or parasite ligands utilized and mechanisms of pathogenesis in these two organs. Exacerbation of pro-inflammatory responses during infection by deletion of the il10 gene resultsin the aggravation of damage to lung and kidney irrespective of the degree of sequestration. The immune response therefore affected both sequestration and histopathology in an organ-specific manner. P. chabaudi AS provides a good model to investigate the influence of the host response on the sequestration and specific organ pathology, which is applicable to human malaria.
Infected red blood cells (iRBC) of many species of the malaria parasite Plasmodium adhere to endothelial cells (EC) in the microvasculature of organs in a process called sequestration. During human Plasmodium falciparum infection iRBC are found in organs such as brain, lungs, spleen, placenta, eyes, subcutaneous fat, heart, bone marrow, gut (reviewed by Haldar et al., 2007), and more rarely in liver (Whitten et al., 2011). Sequestration of P. falciparum is associated with the most severe symptoms of human malaria such as cerebral malaria, acute lung injury (ALI), acute respiratory syndrome (ARDS), and pregnancy malaria (Desai et al., 2007; Haldar et al., 2007).
Other Plasmodium species have also been observed in the microvessels of various organs during infection, including those infecting lower primates (Cox-Singh et al., 2010), rodents (Smith et al., 1982; Gilks et al., 1990; Franke-Fayard et al., 2005; Amante et al., 2010; Claser et al., 2011; Fu et al., 2012), and more recently P. vivax in humans (Anstey et al., 2007; Machado Siqueira et al., 2012). However, only P. falciparum has been found to sequester significantly in the brain, whereas other Plasmodium species have been observed mostly in liver, lung and spleen (Weiss et al., 1986; Gilks et al., 1990; Mota et al., 2000; Franke-Fayard et al., 2005; Anstey et al., 2007; Amante et al., 2010; Claser et al., 2011; Lacerda et al., 2012; Machado Siqueira et al., 2012; Manning et al., 2012).
In addition to sequestration, inflammatory cytokines, such as IFNγ, TNF and IL1, are also associated with severity of P. falciparum and P. vivax malaria (Clark et al., 2008), as well as P. berghei, P. yoelii and P. chabaudi infections of mice (Stevenson and Riley, 2004; Langhorne et al., 2008). Pro-inflammatory molecules can also induce expression of adhesion molecules, such as E-selectin, ICAM-1 and V-CAM1 on the surface of endothelial cells (Aird, 2007a), which increase and strengthen in vitro binding of both P. falciparum and P. vivax iRBCs (Prudhomme et al., 1996; Carvalho et al., 2010). It is thus possible that host inflammatory responses influence both pathology and iRBC sequestration in vivo during infection.
Sequestration in mouse models has mostly been investigated in depth in P. berghei ANKA infections of mice (Lovegrove et al., 2008; Amante et al., 2010; Baptista et al., 2010; Claser et al., 2011; Fonager et al., 2012; Nacer et al., 2012), and to some extent during P. yoelii infections (Martin-Jaular et al., 2011). Studies of P. berghei ANKA have concentrated on sequestration in the brain, often with conflicting results (Franke-Fayard et al., 2005; Baptista et al., 2010). One limitation of studying sequestration in this model is the asynchronous nature of the erythrocytic cycle. P. chabaudi, in contrast, has a synchronous asexual cycle and is the only rodent malaria which demonstrates a defined period of schizont withdrawal from peripheral blood. Parasites have been observed in blood vessels in the liver (Gilks et al., 1990; Mota et al., 2000). Furthermore the inflammatory response to this infection is well characterized (Freitas do Rosario et al., 2012). This infection is thus a very accessible model to examine sequestration, and the influence of parasite-induced inflammation on this process and on malaria pathogenesis.
Here we have carried out a systematic study of sequestration and histopathology of P. chabaudi in C57Bl/6 mice. We show that mature stages of P. chabaudi accumulate early in infection in the spleen, and later actively sequester in the liver and lungs. Histological changes and host cell damage, observed in multiple organs during a primary blood-stage infection, are similar to those observed in P. falciparum and P. vivax malaria in humans. The adaptive immune response and IFNγ signalling increase sequestration and histopathological changes specifically in the liver. By contrast an elevated inflammatory response induced in mice lacking IL-10 (Li et al., 2003; Sanni et al., 2004) increases lung and kidney damage without affecting sequestration in these organs. Plasmodium chabaudi therefore provides an excellent model in which to investigate organ-specific pathology and sequestration in malaria.
Plasmodium chabaudi infected erythrocytes sequester in the lungs and liver in a time-dependent manner
The asexual cycle of P. chabaudi in RBC is completed within 24 h and, uniquely among the rodent malarias, is highly synchronous, enabling determination of the timing of sequestration. Analysis of thin blood films over 8 h from trophozoite to schizont maturation, schizogony, and re-invasion of RBC (ring stages) showed that although mature trophozoites were replaced by ring-stage parasites, fewer than 10% of the parasites were schizonts (Fig. 1A and Fig. S1A) indicating that the majority of mature parasites were absent from peripheral blood. There was a consistent decrease of 20–38% in total parasitaemia prior to the appearance of ring-stage parasites (13.00 h), compared with parasitemia at the earlier stages (9.00 h; Fig. 1B, and Fig. S1B).
Analysis of iRBC in blood vessels of lungs, liver, kidney and brain before (9.00 h), during (12.00 h) and after the withdrawal period (17.00 h, Fig. 1C and D) showed that the decrease in peripheral parasitemia was mirrored by a time-dependent increase in iRBC in lungs and liver during schizogony but not in kidney and brain. At 12.00 h, the most significant accumulation of parasites was observed in the liver, 8 days post infection (p.i.) (peak of infection), where up to 90% of blood vessels contained iRBC, and parasitemia inside the microcapillaries was 2–3 times that of peripheral blood (up to 70%). Of interest the accumulation of parasites in the lungs was only observed in the microcapillaries (Fig. 1C and D) where the parasitemia was significantly increased at 12.00 h compared with that observed in the lungs or in the periphery at 9.00 h. Infected RBCs localized within the microcapillaries were abundant in lung, liver and spleen but were rare in kidney and brain. Strikingly, as shown in representative haematoxylin and eosin (H&E) stained tissue sections and electron micrographs (Fig. 1E and Fig. S2B), iRBC were observed lining the endothelium of liver and lungs. These observations together strongly suggest active sequestration in the organs rather than a result of parasite load, or blockage of the microvessels.
To demonstrate sequestration more directly and to investigate its dynamics we examined organs of mice infected with a transgenic line of P. chabaudi expressing luciferase (PccASluc) (Fig. S3A). The course of the PccASluc infection was similar to that of the wild-type (wt) parasites (Fig. 2A). During schizogony (12.00–14.00 h), PccASluc was imaged in organs of C57BL/6 mice after intracardiac perfusion (Fig. 2B) from day 5–29 p.i., when a detectable parasite recrudescence was observed.
Infected RBCs were predominantly in the spleen early in infection. However, because of the nature of the splenic architecture, perfusion of the spleen is not efficient (Fig. S2A), therefore it was not possible to differentiate between sequestration and accumulation in this organ. By contrast, sequestration could clearly be measured in lungs and liver where perfusion was very effective, and, these were the dominant sites of sequestration by day 8 (Fig. 2C). Interestingly, maximum sequestration in lungs and liver occurred at day 9, when there is a significant host response (Stevenson and Riley, 2004; Freitas do Rosario et al., 2012), and numbers of iRBC in the blood had declined dramatically (Fig. 2C). It is also important to note that the level of sequestration decreases rapidly in the liver after day 9 while it remains relatively high in the lungs until day 13 indicating that the molecules implicated in Pcc AS sequestration are different and/or differentially regulated in these two organs. Only a low level of sequestration was observed in the gut, but luciferase activity could be detected in the surrounding visceral fat tissue (data not shown). Similar to the histological findings, at no time point was sequestration observed in brain or kidney (data not shown).
The adaptive immune response and IFNγ influence sequestration in the liver but not lung during P. chabaudi infection
A P. chabaudi blood-stage infection in C57BL/6 mice induces an immune response characterized by pro-inflammatory cytokines such as TNF and CD4 T-cell derived IFNγ. IL10 is a crucial regulator of this pro-inflammatory response (Li et al., 2003; Sanni et al., 2004; Freitas do Rosario et al., 2012). Pro-inflammatory cytokines can induce or increase expression of some adhesion molecules on the surface of EC (Aird, 2007a), and they influence adhesion of iRBCs to ECs in vitro (Prudhomme et al., 1996; Mota et al., 2000; Carvalho et al., 2010). We therefore asked whether the adaptive immune response and inflammatory cytokines would affect P. chabaudi sequestration in vivo using mice lacking T and B cells (rag1−/−), the IFNγ receptor (ifnγr−/−mice), or IL-10 (il10−/−.) As the major differences in sequestration patterns were seen between days 5 and 9 p.i. in C57BL/6 mice, those time points were chosen for further analysis.
Plasmodium chabaudi iRBCs were still present after perfusion of the organs of rag1−/− mice (Fig. 3B) indicating that adaptive immunity was not required for sequestration. However there was a significant reduction of iRBC sequestration at day 5 (prior to peak infection), and day 9 (1 day post peak infection) in the livers of rag1−/− mice and ifnγr−/− mice, despite higher peripheral parasitemias (Fig. 3A and B). By contrast, deletion of either gene had no effect on sequestration in the lung (Fig. 3B).
The lack of IL10 did not affect overall peripheral parasitemia (Fig. 3A and Li et al., 2003). However there was a significantly greater withdrawal of iRBC from the periphery (Fig. S1D, and Sanni et al., 2004), and, although not statistically significant, the level of sequestration in the liver was, on average, two fold higher in il10−/− mice than in C57BL/6 wt mice (Fig. 3B). The lack of IL10 did not affect sequestration of iRBC in the lungs, which remained at the level of wt mice (Fig. 3B). The accumulation of iRBC in the spleen of il10−/− mice was significantly higher at day 9 compared with wt C57BL/6 mice and rag−/− and ifnγr−/− mice. As it was not possible to differentiate between sequestration and accumulation in this organ, the mechanisms underlying these differences are as yet unknown.
Despite the presence of oedema and haemorrhages previously observed in brains of il10−/− mice (Sanni et al., 2004), there was no observable sequestration of iRBC in this organ (data not shown), suggesting that the pathology associated with the brain is mediated by an inflammatory response.
The effects of the adaptive immune response and pro-inflammatory responses therefore enhance sequestration of P. chabaudi in the liver but not in the lung.
Plasmodium chabaudi infection induces histopathological changes in multiple organs
Histological analysis of brain, lung, liver and kidney was performed throughout the course of the P. chabaudi blood-stage infection in C57BL/6 mice (Fig. 4).
Extramedullary haematopoesis (EMH) and greater numbers of Kuppfer cells were observed in the liver between day 5 and 13 of infection with focal necrosis at peak infection (Fig. 4A). Hepatocellular degeneration and necrosis was indicated by increase in the level of the liver enzyme alanine transaminase (ALT) in plasma on days 8 and 9.
Changes in the lung were observed during acute infection with increased cellularity of the alveolar septae from day 7 to 13 p.i. (Fig. 4B). Flow cytometric analysis showed a significant increase in the number of dendritic cells (CD11c+, MHCIIhigh), monocytes (Ly6Chigh, CD11b+, Ly6G−), neutrophils (Ly6G+, CD11b+, Ly6Cint) and CD8+ T cells, but not CD4 T cells or macrophages (CD11c+, CD11blow/−, F4/80int, MHCII+) in lung tissue (Fig. S4). There were also elevated numbers of IFNγ-producing lymphocytes (Fig. 4B). Of the IFNγ-producing cells, 60% were CD3+CD90.2+, among which 50% were CD4+ and 20% were CD8+ T cells (data not shown). There was a significant increase in the amount of IgM in the BAL (Fig. 4B) indicating some disruption of the alveolar-capillary membrane barrier (Lovegrove et al., 2008) at Day 8, and during the resolution of infection (Day 13).
Tubular dilatation was observed throughout the infection in the kidney showing that P. chabaudi infection induced histopathological changes even in the absence of sequestration (Fig. 4C). However, no significant differences of urea and creatinine levels in serum were observed between infected and uninfected animals at any stage of infection, suggesting no major impairment of kidney function in infected C57Bl/6 mice. There were no observable changes in the brain.
In summary, histopathological changes were observed in liver and lung where sequestration takes place, and some minor changes in the kidney where no sequestration was observed.
Pro-inflammatory responses increase histopathological changes in an organ-specific manner during Plasmodium chabaudi infection
As we had observed that the adaptive response, and pro-inflammatory responses affected sequestration in liver but not lungs, we also asked whether these responses affected any of the P. chabaudi-associated histopathology observed.
In line with the reduction in sequestration described above, hepatocyte damage, as determined by ALT in plasma was significantly reduced in infected rag1−/− mice and ifnγr−/− mice compared with infected wt mice (Fig. 5A). By contrast, the increased ALT levels in il10−/− mice were comparable with those of wt mice (Fig. 5A).
As would be expected, there were few infiltrating cells in the lungs of rag1−/− mice (Fig. S4B) and few IFNγ producing cells compared with wt mice (data not shown), whereas the numbers of these cells were unaltered by the lack of the IFNγ receptor (Fig. 5B). In line with a general increase in the inflammatory response in il10−/− mice (Freitas do Rosario et al., 2012), there were alterations in the populations of infiltrating cells and greater numbers of IFNγ-producing cells in the lungs (Fig. 5B and Fig. S4B) compared with wt mice.
Despite the fact that sequestration was unaltered in the lungs of all of the immunodeficient mice, there was significantly more IgM in the BAL of il10−/− mice compared with infected wt mice (Fig. 5B). Surprisingly there was no change in the amount of IgM in BAL of ifnγr−/− compared with their wt controls, suggesting that IFNγ signalling was not involved in alveolar-capillary membrane barrier disruption. As rag1−/− mice do not produce immunoglobulin it was not possible to use BAL IgM as an indicator of damage to the alveolar-capillary membrane barrier in these mice. Measurement of BAL albumin was similar in wt, ifnγr−/− and rag−/− mice (Fig. S4C) but, consistent with the measurement of IgM, was slightly elevated in infected il10−/− mice.
There was no significant change in kidney histopathology in ifnγr−/− mice, and only a small increase in urea in rag1−/− mice. However, in il-10−/− mice there was clearly more tubular dilatation and significantly elevated urea and creatinine in the plasma, indicating increased kidney damage and loss of function (Fig. 5C).
Adaptive immunity and IFNγ therefore contribute to liver damage, but not to the disruption of alveolar-capillary membrane barrier of the lung, or to damage of any of the other organs examined. The acute lung and kidney injury observed in P. chabaudi infections appear to be independent of adaptive immunity, IFNγ and the amount of parasite sequestration or burden, but are associated with the greater inflammatory response of il10−/− mice.
We have investigated parasite sequestration and histopathological changes occurring in C57BL/6 mice during an infection with the rodent malaria parasite, Plasmodium chabaudi, and show that adaptive immunity and an inflammatory response increase sequestration of P. chabaudi iRBC, and contribute to pathology in an organ-specific manner.
We show, for the first time, sequestration of P. chabaudi iRBC in the lungs during infection and, in agreement with previous histological studies (Cox et al., 1987; Gilks et al., 1990; Mota et al., 2000), demonstrate sequestration in the liver. However no significant sequestration could be observed in kidneys, brain or gut. Although there was not a complete lack of schizonts in peripheral blood, similar to observations of P. vivax infections in humans (Field and Shute, 1956), parasitemia in the microvessels of the lungs and liver was significantly greater than that in peripheral blood during schizogony, and parasites were observed lining the endothelium, strongly supportive of active sequestration in these organs.
There are similarities in this sequestration pattern with other rodent malaria species. Plasmodium berghei ANKA iRBCs are found in the lungs, liver, spleen and visceral fat tissue, but substantially fewer in the brain (Franke-Fayard et al., 2005; Amante et al., 2010; Fonager et al., 2012). P. yoelii has been shown by histology to accumulate in the spleen, liver, brain and kidney (Smith et al., 1982; Fu et al., 2012), but a comparison between organs has not yet been performed. The relative lack of sequestration of the rodent malaria parasites in the brain, but their accumulation in the liver, spleen and lungs is more similar to that described so far for the human infection with P. vivax (De Brito et al., 1969; Anstey et al., 2007; Machado Siqueira et al., 2012; Manning et al., 2012).
Sequestration is mediated by adhesion of parasite proteins expressed on iRBC to EC receptors (reviewed by Rowe et al., 2009). The human parasite P. vivax and rodent malarias lack the var multigene family (Cunningham et al., 2010), which is implicated in sequestration of P. falciparum and associated with cerebral malaria (Rowe et al., 2009; Avril et al., 2012; Claessens et al., 2012). This may be one explanation why, unlike P. falciparum, P. chabaudi and P. vivax do not accumulate in the brain. Although neither the parasite nor the host adhesion molecules involved in its sequestration are known, P. chabaudi shares several analogous multigene families coding for potential parasite adhesion molecules with high similarity to genes of P. vivax (e.g. pir genes) (Cunningham et al., 2010; Lawton et al., 2012). It has been shown in vitro that expression of pir genes from P. vivax in P. falciparum iRBC enhanced their adhesion to EC receptors such as ICAM-1 (Bernabeu et al., 2012). As P. vivax and rodent malaria parasites share this multigene family it is possible that they have a similar potential for cytoadherence by the same host receptors and, thus, explain why they sequester within the same organs.
Histopathological changes occur in several organs during human malaria, some of which are in the organs where iRBC sequestered. Acute lung injury characterized by thickened alveolar septa and intra-alveolar haemorrhages, liver damage associated with Kupffer cell hyperplasia, necrosis and an increase of plasma ALT and bilirubin levels, and kidney failure associated with tubular haemoglobin casts and increased serum creatinine and urea levels are common complications during both P. vivax and P. falciparum infection (Haldar et al., 2007; Anstey et al., 2009). During P. chabaudi infection, we observed similar changes in liver and lung coincident with sequestration such as liver necrosis, with elevated plasma ALT level, and inflammatory cell infiltration in the lung and presence of IgM in the BAL consistent with alveolar-capillary membrane barrier disruption (Lovegrove et al., 2008). We also observed tubular dilatation in the kidney, where no sequestration was observed. The finding of sequestered parasites and tissue damage in the lungs is similar to that described for both P. falciparum and P. vivax (Anstey et al., 2007; Lacerda et al., 2012), and respiratory distress syndromes have been described in all five malaria species infecting humans (reviewed by Taylor et al., 2004). The P. chabaudi experimental model system may therefore facilitate the further investigation of the relationship between sequestered parasites and respiratory clinical syndromes.
The host immune response and particularly pro-inflammatory cytokines can potentially influence both sequestration and pathological sequelae of malaria. In vitro, TNF and IFNγ upregulate expression of endothelial adhesion molecules such as I-CAM, V-CAM, P- and E-selectin (Aird, 2007a), and increase the binding of RBC infected by P. falciparum, P. vivax or P. chabaudi to endothelium in vitro (Prudhomme et al., 1996; Mota et al., 2000; Carvalho et al., 2010). Here we demonstrate that T and B cells and signalling through the IFNγ receptor enhance sequestration of P. chabaudi in the liver but not the lung.
The expression of adhesion molecules on endothelial cells, such as VCAM-1 and ICAM-1, differs both constitutively and after activation in organs such as brain, spleen and lung (Oh et al., 2004; Aird, 2007a,b). Furthermore, P- and E-Selectins, which are highly upregulated in the brain and implicated in cerebral malaria during P. berghei infection, are not expressed on endothelial cells from liver sinusoids (Combes et al., 2004; Aird, 2007a,b). In mice lacking T and B cells or the IFNγ receptor, we observed changes in sequestration only in the liver but not in the spleen or lungs, suggesting that P. chabaudi may use more than one endothelial cell receptor for adhesion, the expression of which may be inducible to different levels in these organs. It is also possible that different populations of iRBC sequester in these organs with different parasite ligands. These possibilities are currently under investigation.
Alteration in inflammatory cytokines also affected the pathological changes observed during Plasmodium chabaudi infection but differently in the different organs. The lack of the IFNγ receptor and T and B cells resulted in a reduction not only in liver sequestration but also lower ALT levels in the plasma, despite a higher parasitemia. In il10−/− mice with an exacerbated inflammatory response to P. chabaudi infection (Sanni et al., 2004; Freitas do Rosario et al., 2012), there was increased IgM and albumin in the BAL indicating disruption of the alveolar-capillary membrane barrier, despite no change in parasitemia or amount of sequestration in the lungs. Kidney damage was also observed in these mice without any significant parasite sequestration. Similarly, there was no sequestration in the brain, where oedema and haemorrhages have been described in P. chabaudi infected il10−/− mice (Sanni et al., 2004).
Our results demonstrate that adaptive immunity and inflammatory responses affect sequestration and histopathology in an organ-specific manner. To understand fully the pathogenesis of human malaria and the host/pathogen interactions taking place during this infection, it will therefore be necessary to dissect these mechanisms in all organs of interest. P. chabaudi is a good model in which to dissect these mechanisms, which may be directly relevant for aspects of both P. falciparum- and P. vivax associated disease.
This study was carried out in accordance with the UK Animals (Scientific Procedures) Act 1986 (Home Office licence 80/2538), and was approved by the National Institute for Medical Research Ethical Committee. All surgery was performed under sodium pentobarbital anaesthesia, and all efforts were made to minimize suffering.
Female C57BL/6 and BALB/c, B6.rag1−/− (Mombaerts et al., 1992), B6.ifnγr−/− (Huang et al., 1993), B6.il10−/− (Kuhn et al., 1993) aged 6–8 weeks from the SPF unit at the MRC National Institute for Medical Research were housed conventionally with sterile bedding, food and irradiated water on a reverse 12 h light–dark cycle.
A transgenic line of Plasmodium chabaudi chabaudi (AS) expressing luciferase (PccASluc) under the control of the constitutive promoter EF1α was generated by transfection with the plasmid pPc-LUCCAM targeting the small subunit ribosomal RNA locus and the insertion was verified by Southern blot analysis as described (Spence et al., 2011) (Fig. S3A). For all other experiments, a cloned line of wild type P. chabaudi AS strain was used in this study. Infections were initiated by intraperitoneal (i.p.) injection of 105 iRBC, and monitored by examination of Giemsa-stained blood films. PccASluc parasites were maintained in mice given acidified water containing pyrimethamine to select for transfected parasites (Spence et al., 2011).
Mice were euthanized by i.p. injection of pentoject (Animal Care), the trachea was cannulated and lungs inflated by injection of 3 ml of 4% M/V formaldehyde (VWR). The brain, left lobes of the lung and liver, left kidney and spleen from infected and uninfected mice were fixed in 4% VWR for 24 h at room temperature. Fixed organs were embedded in wax, sectioned (5 μm), and stained with H&E. Sections were examined microscopically and changes recorded using a standard non-linear semi-quantitative scoring system using a scale from 0 to 5 adapted from Shackelford et al. (2002) (Scudamore, in press). Significant findings were scored 0 (where no change was detectable), 1 when the least amount of change was detectable by light microscopy (usually < 10% of tissue affected), 2 when change was readily detected but not a major feature (< 20%), 3 when the change was more extensive and might be expected to correlate with changes in organ weight or function, 4 when up to 75% of tissue was affected by the change and 5 when the whole tissue was affected by a change which was likely to be functionally relevant. Organs from infected mice were always compared with those from uninfected controls on the same day. The percentage of vessels in each organ containing iRBC was determined from 400 vessels, and the percentage parasitemia in the vessels was counted in a minimum of 200 RBC.
In vivo imaging and luciferase assay
At selected times after infection with PccASluc, at maximum sequestration (12.00 h to 14.00 h, reverse light, Fig. 1), d-luciferin (150 mg kg−1, Caliper Life Sciences) was injected subcutaneously 5 min before imaging. Mice were terminally anaesthetized and perfused by intracardiac injection of PBS (Fig. S2A). The brain, lungs, liver, spleen, left kidney and gut were removed immediately and luciferase was assessed using in vivo Imaging System IVIS Lumina (Xenogen), with a 10 cm field of view, a binning factor of 4, and an exposure time of 10 s. Bioluminescence (p/s) was quantified with the software Living Image (Xenogen) by adjusting a region of interest to the shape of each organ.
To account for the influence of total parasite load on the number of parasites sequestered in the organs, bioluminescence in the organs was normalized to total parasite load. Because of the dark pigmentation of C57BL/6 mice, measurement of parasite burden by whole body imaging was not accurate (Fig. S3B). For each mouse, 2 μl of heparinized tail blood was collected before sequestration (9.00 h). Bioluminescence was assessed with the Luciferase Assay System (Promega) according to the manufacturer's protocol and quantified with the TECAN Safire2 plate reader and Magellan software (Tecan). Under these conditions, bioluminescence intensity is proportional to the amount of parasites in this blood volume (Fig. S3C). This value was taken to reflect parasite burden in the whole body. Luciferase activities measured in the organs were normalized to this value, allowing comparison between mice with different parasite burdens.
Alanine transaminase, urea and creatinine quantification in the plasma
Alanine transaminase, urea and creatinine were quantified in plasma from blood obtained by cardiac puncture under terminal anaesthesia using a Cobas C111 chemistry analyser (Roche).
Bronchoalveolar lavage fluids (BAL) analysis
Uninfected and infected mice were euthanized by intraperitoneal injection of pentoject (Animal Care). The trachea was cannulated and bronchoalveolar lavage (BAL) performed using 1 ml of PBS (Gibco/Invitrogen). BALs were centrifuged at 900 g for 5 min at 4°C. IgM and albumin in BAL were measured by sandwich ELISAs (IgM, eBioscience, San Diego CA; albumin, Genway San Diego, CA).
Flow cytometric analysis
After BAL, lung tissue was dissected, minced, incubated in Liberase (40 μg ml−1; Roche Diagnostics) at 37°C for 1 h and disrupted by subsequent passage through a 70 μm nylon cell strainer (BD Bioscience). Cells were counted, and diluted in Iscove's modified Dulbecco's medium (Sigma) supplemented with 10% FBS (PAA), 0.05 mM β-mercaptoethanol (Gibco), 2 mM l-glutamine (Gibco), 0.5 mM sodium pyruvate (Sigma), 6 mM Hepes (Gibco), 100 U ml−1 penicillin and 100 μg ml−1 streptomycin (Gibco). For flow cytometry, single-cell suspensions were incubated with FcR block (BD Bioscience) followed by specific Abs: CD4-V500 (BD Bioscience), Ly6G-PE (Biolegend), MHCII-FITC (Biolegend), 7AAD-PerCP (eBiosciences), Ly6G-V500 (Biolegend), CD90.2-APC (Biolegend), CD11c-APC-Cy7 (Biolegend), CD11b-PacificBlue (eBiosciences), F4/80-PE-Cy7 (Biolegend), CD19-APC-Cy7 (Biolegend), CD3-PE-Cy7 (Biolegend). After surface labelling, cells were fixed with 2% paraformaldehyde in PBS, and permeabilized with Cytofix/CytopermTM (BD Biosciences). Intracellular IFNγ was detected using IFNγ-PE (Biolegend) as described (Freitas do Rosario et al., 2012). Samples were acquired on a FacsCanto II® (BD Bioscience) using Summit Cytomation FlowJo (Tree Star, Inc.) software for analysis.
Transmission electron microscopy
Mice were terminally anaesthetized and perfused by intracardiac injection of PBS. Samples were immersion fixed in 2% glutaraldehyde/2% paraformaldehyde and post fixed in 1% osmium tetroxide using 0.1 M sodium cacodylate buffer pH 7.2. Aqueous uranyl acetate was followed by dehydration through a graded ethanol series, propylene oxide and embedding was in Epon, 50 nm sections were mounted on pioloform coated grids and stained with ethanolic uranyl acetate followed by Reynold's lead citrate. They were viewed with Gatan Orius 1000 CCD.
Data are shown as means and SEM. The non-parametric Mann–Whitney U test was used and P values below 0.05 were considered as statistically significant.
We would like to thank NIMR Biological Services, the Flow Cytometry facilities, Radma Mahmood of the Histology facilities and Liz Hirst of the Electron Microscopy facilities for their skilled technical assistance. This work was supported by the Medical Research Council, UK (U117584248); Singapore A*Star-UK MRC collaborative grant (A*Star reference 10/1/22/24/630), and received funding from the European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement no. 242095-EVIMalaR. P.J.S is the recipient of a Leverhulme Trust early career fellowship.
Conflict of interest disclosures
The authors declare that they have no competing interests.