Intravital microscopy enabled us to observe and document the cascade of events leading to malaria sporozoite infection of the liver. Our recordings show that sporozoites entering the liver lobule are abruptly arrested by binding to the sinusoidal cell layer, presumably by recognizing liver-specific ECM proteoglycans protruding through the endothelial fenestration into the sinusoidal lumen [4
]. Sporozoites then glide along the sinusoid until they encounter a Kupffer cell, where they stop with their apical end attached to the phagocyte. After a pause, they push slowly across the sinusoidal cell barrier, showing a point of constriction in their outline. Once inside the liver parenchyma, they continue to migrate for many minutes, fatally wounding several hepatocytes in the process. Eventually, the parasites settle down in one final hepatocyte, become surrounded by a parasitophorous vacuole membrane, and differentiate to EEFs (model in ).
Model of Plasmodium Sporozoite Infection of the Mammalian Liver
The overall velocities of sinusoidal gliding and hepatocyte transmigration are similar to sporozoite gliding in Matrigel (approximately 1.8 μm/s; see ), on artificial surfaces in vitro (1–3 μm/s) [34
], and in mosquito salivary glands (≤ 2 μm/s) [27
], but migration through Kupffer cells is slower by an order of magnitude. The speeds of sinusoidal gliding and hepatocyte transmigration () fall into the general speed range of 1–10 μm/s found for various other apicomplexan parasites [35
], including Toxoplasma gondii
], Eimeria tenella
], and Gregarina polymorpha
]. Interestingly, the velocity of Plasmodium
ookinetes (0.08–0.25 μm/s) is slower than that of sporozoites [39
] and similar to that of sporozoites traversing Kupffer cells, perhaps reflecting an adaptation to the environment of the mosquito midgut.
Gliding Speeds of Apicomplexan Parasites
The process of Kupffer cell passage differs from passage through hepatocytes by more than speed. Entry into hepatocytes does not involve a pause before entry, a constriction in the parasite during passage, or a change in migration speed (see ). Perhaps the slower speed relates to the need to form a vacuole or duct for passage through Kupffer cells [14
] in contrast to direct breaching of the cell membrane during transmigration through hepatocytes [25
]. Alternatively, the barrier that causes the constriction and slows the parasites could be the ECM in the space of Disse just beyond the Kupffer cell. A similar phenomenon has been observed for P. gallinaceum
ookinetes, which cross the microvilli-associated network of Aedes aegypti
midgut epithelia at roughly 10-fold slower a speed compared with the subsequent migration on or through the cells [40
]. However, since host cell entry can also cause the formation of a constriction in apicomplexan parasites [44−46], additional work is required to identify the nature of the barrier presented to sporozoites leaving the sinusoid.
In contrast to motility on artificial surfaces in vitro, where sporozoites maintain a fixed crescent shape and move in circles or spirals [34
], sporozoite migration in vivo and in Matrigel is characterized by a more linear migration pattern, high parasite flexibility, and frequent changes in direction, most likely guided by structural tissue components. The general movement pattern in the liver resembles that of sporozoites gliding in the skin after transmission by mosquito bite [1
]. P. berghei
sporozoites in An. stephensi
salivary glands also move according to tissue architecture and follow the outline of the acinar epithelia and the secretory duct [27
]. It appears that Plasmodium
sporozoites, in general, are guided by the three-dimensional arrangement of matrix structures to reach their next destination in a tissue.
We were surprised to find that P. berghei
sporozoites traverse a similar number of hepatocytes in livers from mice and rats. P. berghei
is much less infectious to mice than to its natural host, the African wild rat Thamnomys surdaster
], and induces a more pronounced inflammatory reaction in these rodents, while young rats exhibit intermediate levels of infection and inflammation [50
]. Based on our data, it appears that sporozoite transmigration is an intrinsic feature of Plasmodium
sporozoites, which occurs irrespective of the host–parasite combination. Thus, it may be the degree of the mismatch between parasite and host that determines the extent of the inflammatory response to hepatocyte necrosis.
Malaria sporozoite migration can cause significant injury to the liver tissue, as demonstrated by the inoculation-size-dependent increase in the serum ALT levels for at least 2 d after intravenous inoculation of P. yoelii
sporozoites (see Video S12
). Uninfected gland extracts had a significant, albeit temporary, effect, suggesting that the sustained ALT elevation observed 24 and 52 h after sporozoite inoculation was indeed a consequence of parasite migration. Although the ALT levels measured here are well below those observed in experimental fulminant hepatitis caused by murine hepatitis virus 3 infection [53
], the sporozoite-induced damage was large enough to induce deposition of detectable amounts of collagen in the space of Disse, focal Kupffer cell hyperplasia, and groups of proliferating hepatocytes at later time points. The liver damage induced by bite of infected mosquitoes, although morphologically detectable, remained below the detection limit of the ALT assay, most likely because the number of sporozoites reaching the liver under natural transmission conditions is too low to induce a significant hepatotoxic effect. Mosquitoes generally transmit less than 100 sporozoites per bite [1
], so under optimal conditions 150 mosquitoes would have transmitted roughly 15,000 sporozoites per mouse, only part of which leave the skin and travel to the liver (J. Vanderberg and U. Frevert, unpublished data). These findings also suggest either that mosquitoes do not inject significant amounts of saliva into the bloodstream while probing the skin or that saliva has no adverse effect on the liver parenchyma. In conclusion, while experimental infection with large numbers of purified sporozoites can inflict measurable liver injury, natural infection by mosquito bite poses no risk to liver function, even when small rodents are exposed to large numbers of well-infected mosquitoes under laboratory conditions. These data confirm that sporozoite-induced liver injury is of no concern to malaria-infected individuals living in endemic areas.
A contradictory model of sporozoite entry into the liver was recently presented by Yuda and colleagues [23
]. The group generated P. berghei
parasites deficient in the micronemal proteins SPECT1 and SPECT2, which exhibit greatly diminished liver infectivity after intravenous inoculation into mice. Experimental data on the function of the SPECT proteins are lacking to date, but SPECT2 has been proposed to contain a membrane attack complex that mediates membrane wounding and, consequently, sporozoite transmigration through cells. In vitro results obtained from permanent nonphagocytic cell lines supported this hypothesis, but the authors then extrapolated from these data to Kupffer cells, hypothesizing that SPECT mutants have lost their ability to wound the Kupffer cell membrane and therefore cannot pass through the cytoplasm of these phagocytes. This is in contrast to Kupffer cell passage by membrane invagination as documented by others [14
]. To explain this discrepancy and taking into account the putative membrane attack complex in SPECT2, one could speculate that SPECT mutants accumulate inside Kupffer cells because they have lost the ability to escape the vacuole surrounding them in these cells.
In another study by Ishino and colleagues, Kupffer cell elimination more than doubled the rate of liver infection by P. berghei
]; this would not be possible at the approximately 80% infection rate presented here. The most likely explanation for this discrepancy is that our results are based on natural sporozoite transmission by mosquito bite, while Ishino and colleagues used intravenous injection, which results in markedly lower liver infection efficiencies relative to the size of the inoculum [56
]. It is generally believed that preparations of purified sporozoites contain a considerable percentage of noninfectious parasites, the majority of which is likely cleared from the bloodstream by Kupffer cells. Kupffer cells may become activated upon phagocytosis of dead sporozoites, and the resulting inflammatory microenvironment would clearly inhibit EEF development [57
]. This would not occur in the absence of Kupffer cells [58
], thus explaining the large increase in the liver infection rate in clodronate-treated mice. In addition, clodronate released by leakage from liposomes or by dying Kupffer cells has been suggested to suppress inflammatory cytokines [58
], which would also enhance EEF survival.
sporozoites continuously release vesicles covered with CSP and thrombospondin-related adhesive protein from their cell surface membrane [47
]. When gliding on artificial surfaces such as glass, sporozoites leave these vesicles behind in the shape of a trail, but when migrating on cultured cells or in tissues such as mosquito salivary glands, the released CSP translocates across membranes; it initially spreads across the cytoplasm of the affected cells and inhibits protein synthesis and later redistributes to the perinuclear space [62
]. Since sporozoites release CSP into Kupffer cells in vitro [14
], we expect that this also occurs with liver endothelia and Kupffer cells in vivo. The significance of this is that both sinusoidal endothelia and Kupffer cells are fully mature antigen-presenting cells that express major histocompatibility complex class I and II molecules as well as the co-stimulatory molecules CD80 and CD86, they are able to prime naïve CD4+
T cells in vitro, and they can cross-present alloantigen to CD8+
T cells [64
]. Intimate contact between sporozoites and nonparenchymal cells could be expected to lead to the presentation of parasite antigen—in particular, CSP-derived peptides. However, due to its ribotoxic effect, translocated CSP may interfere with antigen processing. In addition, the unique microenvironment of the liver generally favors the development of tolerance rather than inflammation and immunity, a property thought to have evolved to avoid overreaction to the continuous influx into the portal circulation of foreign materials such as bacteria and endotoxins from the intestines [68
]. Portal vein tolerance [70
] is predominantly mediated by Kupffer cells [71
], can occur irrespective of nature and origin of the antigen and has been implicated in the acceptance of liver allografts, the reduced rejection of allografts when preceded by portal venous administration of donor antigen, the high frequency of tumor metastases in the liver, and the persistence of intrahepatic pathogens such as hepatitis C and hepatitis B virus [72
]. Does this unusual route of entry, together with translocation of CSP by migrating sporozoites, enable malaria sporozoites to take advantage of the unique tolerogenic nature of the liver? Can Plasmodium,
one of the deadliest infectious agents worldwide, avoid the generation of an effective immune response against the continuous influx of sporozoites in endemic areas so successfully because it begins its life cycle in the mammalian host with a round of multiplication in the liver? The liver may be the preferred site for multiplication because of its unique nature as an immune organ as opposed to other tissues that could have possibly supported replication but do not possess this specialized property.