Abstract
ABSTRACTHomeostasis of mammalian cell function strictly depends on balancing oxygen exposure to maintain energy metabolism without producing excessive reactive oxygen species. In vivo, cells in different tissues are exposed to a wide range of oxygen concentrations, and yet in vitro models almost exclusively expose cultured cells to higher, atmospheric oxygen levels. Existing models of liver-stage malaria that utilize primary human hepatocytes typically exhibit low in vitro infection efficiencies, possibly due to missing microenvironmental support signals. One cue that could influence the infection capacity of cultured human hepatocytes is the dissolved oxygen concentration. We developed a microscale human liver platform comprised of precisely patterned primary human hepatocytes and nonparenchymal cells to model liver-stage malaria, but the oxygen concentrations are typically higher in the in vitro liver platform than anywhere along the hepatic sinusoid. Indeed, we observed that liver-stage Plasmodium parasite development in vivo correlates with hepatic sinusoidal oxygen gradients. Therefore, we hypothesized that in vitro liver-stage malaria infection efficiencies might improve under hypoxia. Using the infection of micropatterned co-cultures with Plasmodium berghei, Plasmodium yoelii or Plasmodium falciparum as a model, we observed that ambient hypoxia resulted in increased survival of exo-erythrocytic forms (EEFs) in hepatocytes and improved parasite development in a subset of surviving EEFs, based on EEF size. Further, the effective cell surface oxygen tensions (pO2) experienced by the hepatocytes, as predicted by a mathematical model, were systematically perturbed by varying culture parameters such as hepatocyte density and height of the medium, uncovering an optimal cell surface pO2 to maximize the number of mature EEFs. Initial mechanistic experiments revealed that treatment of primary human hepatocytes with the hypoxia mimetic, cobalt(II) chloride, as well as a HIF-1α activator, dimethyloxalylglycine, also enhance P. berghei infection, suggesting that the effect of hypoxia on infection is mediated in part by host-dependent HIF-1α mechanisms.
Highlights
Malaria affects 250 million people and causes approximately a million deaths each year (World Health Organization, 2011)
They show that the exposure of micropatterned co-cultures of primary human hepatocytes and supporting stromal cells to different levels of oxygen leads to profound changes in malaria infection efficiency and parasite development
We have shown that the cell surface oxygen concentration experienced by primary adult human hepatocytes in vitro influences their ability to support a productive liver-stage malaria infection by P. berghei, P. yoelii and P. falciparum
Summary
Malaria affects 250 million people and causes approximately a million deaths each year (World Health Organization, 2011). Micropatterned co-cultures (MPCCs) of primary human hepatocytes (PHHs) and supporting stromal fibroblasts result in stable hepatocyte function, including albumin secretion, urea production and cytochrome P450 levels, for several weeks compared with hepatocytes alone (Khetani and Bhatia, 2008) Another feature of the in vivo hepatic microenvironment is the presence of a range of oxygen tensions (Wölfle et al, 1983), which is thought to be a factor that contributes to hepatic zonation, a compartmentalization of functions along the axis of perfusion (Jungermann and Kietzmann, 1996; Jungermann and Kietzmann, 2000). In vitro liver-stage malaria culture platforms might be improved by altering microenvironmental oxygen concentrations
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