LAP-like process as an immune mechanism downstream of IFN-γ in control of the human malariaPlasmodium vivaxliver stage

Abstract

IFN-γ is a major regulator of immune functions and has been shown to induce liver-stage Plasmodium elimination both in vitro and in vivo. The molecular mechanism responsible for the restriction of liver-stage Plasmodium downstream of IFN-γ remains uncertain, however. Autophagy, a newly described immune defense mechanism, was recently identified as a downstream pathway activated in response to IFN-γ in the control of intracellular infections. We thus hypothesized that the killing of liver-stage malarial parasites by IFN-γ involves autophagy induction. Our results show that whereas IFN-γ treatment of human hepatocytes activates autophagy, the IFN-γ-mediated restriction of liver-stage Plasmodium vivax depends only on the downstream autophagy-related proteins Beclin 1, PI3K, and ATG5, but not on the upstream autophagy-initiating protein ULK1. In addition, IFN-γ enhanced the recruitment of LC3 onto the parasitophorous vacuole membrane (PVM) and increased the colocalization of lysosomal vesicles with P. vivax compartments. Taken together, these data indicate that IFN-γ mediates the control of liver-stage P. vivax by inducing a noncanonical autophagy pathway resembling that of LC3-associated phagocytosis, in which direct decoration of the PVM with LC3 promotes the fusion of P. vivax compartments with lysosomes and subsequent killing of the pathogen. Understanding the hepatocyte response to IFN-γ during Plasmodium infection and the roles of autophagy-related proteins may provide an urgently needed alternative strategy for the elimination of this human malaria.autophagy | LC3-associated phagocytosis | IFN-γ | malaria S everal hundred million cases of human malaria are reported annually, and nearly 600,000 people die from the disease each year (1). Of the five species that infect humans, Plasmodium vivax is not only the most geographically widespread, but also the most prevalent malarial parasite in areas outside Africa. As such, it has caused massive morbidity in these regions of the world. Although malaria caused by P. vivax was previously regarded as benign compared with that caused by Plasmodium falciparum, the recent alarming increase in both the severity and the drug resistance of P. vivax infection has raised concern (2).The widespread distribution of P. vivax has been attributed to the parasite's ability to remain dormant in the liver for years before reactivation (3). The molecular mechanism responsible for P. vivax dormancy is unknown, and knowledge of Plasmodiumhepatocyte interactions remains very limited. Nonetheless, because the number of liver-stage parasites is in the range of 100, whereas in the blood stage as many as 10 13 organisms may be found (4), intervention at the liver stage would seem to offer a better strategy for parasite elimination. A prerequisite to this route of malaria control and the development of novel therapies is a better understanding of liver-stage Plasmodium and its interactions with host hepatocytes. IFN-γ was previously shown to exhibit antimalarial activity against the liver stag...

IFN-γ protects hepatocytes againstPlasmodium vivaxinfection via LAP-like degradation of sporozoites

“…Despite these experimental difficulties, in PNAS, Boonhok et al (1) use a membrane feeding system with freshly isolated blood from P. vivax patients to infect mosquitoes and obtain sporozoites for study in a specially designed human hepatocyte cell line. With this, Boonhok et al (1) provide an essential piece of information on the P. vivax and hepatocyte interaction by demonstrating that liver cells stimulated with interferon gamma (IFN-γ) can mount an intracellular immune response against P. vivax sporozoites that largely depends on a novel form of noncanonical autophagy. This mechanism is shown to be similar to microtubule-associated protein light chain 3 (LC3)-associated phagocytosis (LAP), a process in which LC3 is deposited directly on the pathogen-containing vacuole, leading to a formation of single-membrane phagosome with final lysosomal fusion and degradation of the pathogen (Fig.…”

“…However, the mechanisms by which IFN-γ acts to suppress parasite growth are unclear. Using their in vitro human hepatocyte infection system, Boonhok et al (1) demonstrate that IFN-γ treatment of hepatocytes results in clearance of ∼50% of intracellular P. vivax sporozoites without host cell death. Importantly, this demonstrates that IFN-γ can trigger an intracellular defense mechanism against P. vivax parasites that is independent of immune effector cells.…”

“…Both canonical and noncanonical autophagy require Beclin-1, PI3K, and Atg5, whereas ULK1 is only required for the canonical autophagy pathway. Boonhok et al (1) demonstrate that inhibition of Beclin-1, PI3K, or Atg5 results in ∼50% less IFN-γ-triggered parasite clearance, whereas inhibition of ULK1 had no effect, suggesting that the clearance mechanism largely depends on a noncanonical autophagy pathway, similar to LAP, with the exception that sporozoites are not phagocytized but rather invade the hepatocyte. They further validated this finding through microscopy studies showing LC3 deposition on the parasitophorous vacuole, and colocalization of parasites with Lysotracker red (LTR) stained acidic vesicles in response to IFN-γ stimulation.…”

“…In LAP, phagocytosis is followed by deposition of LC3 onto the phagosomal membrane, a process dependent on Beclin 1, PI3K, and Atg5, but not requiring ULK1. In human hepatocytes stimulated with IFN-γ, Boonhok et al (1) demonstrate that a LAP-like process occurs when LC3 is deposited onto the PVM of sporozoites, followed by lysosomal fusion and degradation. This differs from a previously described autophagic pathway, with similarities to selective autophagy, described for P. berghei and P. yoelii sporozoites (5,6).…”

“…Pathogen neutralization can then occur by autophagosome engulfment or fusion with pathogen vacuoles, followed by lysosomal fusion, direct lysosomal fusion to pathogen vacuoles mediated by autophagy proteins, or lysosome-independent pathogen vacuole membrane destruction mediated by autophagy proteins, such as described for Toxoplasma gondii (15,16). Boonhok et al (1) now provide the first evidence, to our knowledge, of a unique noncanonical autophagic pathway for the clearance of the human pathogen, P. vivax, providing important information about the host−pathogen interaction in malaria.…”