Visualization of parasite dynamics in the host tissues: application of tissue transparency technology to parasite research

Abstract

Many parasite species migrate to another site of infection after entering the host body. Such parasite dynamics are closely related to pathogenicity, but it is not easy to observe such parasite behavior deep within the organs. In recent years, technology that can make organs transparent has been developed that enables us to observe deep within organs ex vivo while maintaining their three-dimensional structure. This review describes a series of attempts to apply this technology to understand the behavior of Toxoplasma gondii in the host body. A series of studies has shown that T. gondii tachyzoites that infect leukocytes can reach target organs far from the site of invasion via the circulatory system. In addition, infected leukocytes in the bloodstream adhere more readily to vascular endothelial cells than uninfected leukocytes and are more likely to remain inside the target organs. When infected leukocytes adhere to the vascular endothelial cells of the target organ, the tachyzoites inside the cells immediately escape and infect the parenchyma of the organs. As described above, organ transparency technology is a powerful tool for understanding the internal dynamics of parasites.

Parasites of many species, whether helminths or protozoans, migrate from the site of invasion through the host body to reach target organs. Parasitic dynamics in the host body are essential for successful infection by the parasite and are closely related to pathogenicity. For example, the larvae of Mesocestoides vogae, a cestode belonging to the family Mesocestoididae, behaves differently in the body of the terminal host, the canine, at different sites of infection. Its larvae, tetrathyridium, are approximately 1 mm long and orally infective to the definitive host. Tetrathyridia in the intestinal tract differentiate into adult worms without causing severe symptoms [23]. In contrast, some tetrathyridia migrate through the intestinal wall, invade the peritoneal cavity, and proliferate asexually. Proliferation in the peritoneal cavity results in refractory peritonitis [26]. When humans are infested with eggs of Taenia solium, numerous larval cysts infect systemic tissues. The larval cysts that form in the subcutis and muscles do not cause severe symptoms, but those that form in the brain cause severe neurological symptoms and are sometimes fatal [15]. Thus, the site of parasite infection in the host body is closely related to pathogenesis and the progression of the parasite’s life cycle. Understanding the characteristics of parasite migration within the host body is essential for appreciating the survival strategies and pathogenicity of many parasite species.

Toxoplasma gondii is a parasite that infects a particularly diverse range of organs and tissues and causes a variety of pathological conditions. T. gondii, an intracellular parasitic protozoan, enters the host body through the intestinal tract following the oral ingestion of cysts or oocysts; however, gastrointestinal symptoms are not the only primary symptoms of toxoplasmosis. In pigs, eye and nasal discharge, respiratory disease, cyanosis, and neurological symptoms are often observed in young individuals [21]. Reproductive disorders have also been reported in goat and sheep infected with T. gondii [21]. In humans, encephalitis, abortion or stillbirth, and retinochoroiditis are common symptoms of toxoplasmosis [18]. These findings indicate that T. gondii parasites that invade the intestinal tract migrate to the respiratory tract, nervous system (including the eyes), and reproductive organs, causing disease. This manuscript reviews a series of studies on the behavior of parasites in the host body, focusing on T. gondii, a protozoan that causes zoonotic diseases.

LOW TARGET ORGAN/CELL TYPE SPECIFICITY OF T. GONDII AND DIVERSE TOXOPLASMOSIS PATHOGENESIS

Toxoplasma gondii has a wide host range and infects almost all mammals and birds. The target organ/cell-type specificity of T. gondii is also low, with the parasite infecting almost all organs, including the brain, placenta, muscles, and lungs. Therefore, the symptoms produced in toxoplasmosis are very diverse, depending on which organs and tissues the parasite infects. The type of pneumonia caused by growth of the parasite in the lungs is seen in both humans and animals, but cases are more common in pigs [2, 7, 16]. Retinochoroiditis caused by the parasite crossing the blood-retinal barrier and entering the eye is another typical symptom of toxoplasmosis [2, 3]. Placentitis and encephalitis caused by the parasite crossing the blood-placenta barrier and blood-brain barrier, respectively, are also common symptoms of toxoplasmosis [2, 12, 17]. Thus, T. gondii has the characteristic that it can cause completely different pathologies, depending on the site of infection. Therefore, to understand the pathogenesis of toxoplasmosis, it is necessary to understand how tachyzoites reach various organs and invade their target cells. In particular, the potential mechanisms by which they cross biological barriers, such as the blood-brain and blood-placental barriers, are important to elucidate.

LONG-DISTANCE MIGRATION OF T. GONDII FROM THE INITIAL INFECTION SITE TO AROUND THE HOST BODY

During the early phase of T. gondii infection, tachyzoites disseminate from the initial site of infection, the small intestine, to sites throughout the whole body and invade a variety of organs. During this process, the tachyzoites travel long distances over a short period of time. As tachyzoites are transiently detected in the blood of infected animals a few days after infection, they must disseminate through the host body via the circulatory system. When we observed the blood of infected mice during this period, we found both tachyzoite-infected leukocytes (intracellular tachyzoites) and free extracellular tachyzoites in the plasma [25]. It is common practice to inoculate mice intravenously with large quantities of purified tachyzoites in experimental infection protocols, and it is clear that extracellular tachyzoites have certain infectivity. However, it is still controversial as to which is the main dissemination route into the peripheral tissues.

To answer this question, we established two transgenic parasites expressing green fluorescent protein (green tachyzoite) and red fluorescent protein (red tachyzoite). Then, green and red tachyzoites were prepared as intracellular and extracellular forms, respectively, and injected simultaneously into the tail vein of a mouse (Fig. 1A). After a few days, many green tachyzoites were found in the lungs, the spleen, liver, and brain. In contrast, only a few red tachyzoites were observed in these organs (Fig. 1A) [25]. When green and red tachyzoites were prepared as the opposite forms and injected, the majority of tachyzoites in these tissues had the red intracellular type color. This indicated that tachyzoites present within leukocytes substantially contributed to the transmission of tachyzoites via the host circulatory system. Subsequently, various research groups have scrutinized the behavior of tachyzoite-infected cells by cell type, including neutrophils, monocytes, and dendritic cells, to determine the contribution of each to the “long-distance movement” of T. gondii tachyzoites [4, 9, 20]. For example, it has been reported that CD11b⁺ and CD11c⁺ monocytes in the bloodstream play important roles in the transfer of T. gondii from the bloodstream to the brain in mice [5].

Fig. 1.

Visualization of T. gondii in mouse tissues. (A) Schematic diagram of an experiment comparing the ability of intracellular and extracellular tachyzoites in the bloodstream to infect target organs. (B) An example of transparency-treated tissue. Shown here is a transparency-treated mouse fetus. (C) Schematic diagram showing the vascular endothelial adhesive capacity of infected and uninfected leukocytes in the bloodstream. Green indicates leukocytes and red indicates tachyzoites. The inset shows leukocytes observed inside a transparency-treated lung. The red structures inside the leukocytes in the elongated form, indicated by arrowheads, are tachyzoites. Figure from cited reference [1], quoted with modifications. (D) Schematics of the Trojan Horse Hypothesis and the Hitchhiker’s Hypothesis. (E) Changes over time of infected leukocytes co-cultured on monophasic sheets of vascular endothelial cells. Leukocytes in green and tachyzoites in red. Arrows indicate a single tachyzoite that egressed from inside the leukocyte during observation. Figure from cited reference [1], quoted with modifications.

RETENTION OF T. GONDII TACHYZOITE-INFECTED LEUKOCYTES IN THE TARGET ORGAN

The next question to be asked is how do the tachyzoite-infected leukocytes that flow into the systemic circulation reach their target organs? To address this issue, we developed a real-time quantitative PCR (qPCR) method to determine the rate of infection of peripheral blood mononuclear cells (PBMCs) in the blood and solid organs of mice [24]. Tachyzoite-infected PBMCs were injected into mouse tail veins, and the infection rates of PBMCs remaining in the lungs and liver were examined using qPCR. PBMCs inside the lungs and liver showed significantly higher infection rates than the PBMCs originally inoculated into the veins [24]. This suggested that tachyzoite-infected PBMCs in the general circulation remained in the solid organs more effectively than the non-infected PBMCs. To confirm this, we used tissue transparency techniques (Fig. 1B) to visually observe the behavior of infected PBMCs within organs. Since the 2010s, attempts have been made to provide the transparent visualization of organs and tissues of living organisms [14, 22]. These techniques have made it possible to remove organs from experimentally infected animals and easily observe their interiors while preserving their three-dimensional structure. There are various techniques for achieving tissue transparency, but some cause morphological changes as the tissue expands or atrophies during the transparency process. Additionally, these techniques are not necessarily suitable for the observation of intracellular protozoan parasites in single cells. However, in 2013, a new method was developed that caused almost no change in tissue morphology [10], and we found that it was possible to observe tachyzoites infecting the interior of host cells located deep within the tissue using this method [1]. Leveraging this technique to observe the behavior of leukocytes and tachyzoites entering the lungs from the systemic circulation, we found the following [1]; (1) over half of the infected leukocytes in the lungs appeared elongated in shape, suggesting they were adhered to solid tissue; (2) most of the uninfected leukocytes in the lungs maintained a rounded shape; and (3) the number of elongated infected leukocytes increased within a few hours of leukocyte influx into the lungs. These results indicated that tachyzoite-infected leukocytes adhered to solid tissue, probably to vascular endothelial cells, more effectively than non-infected leukocytes and thereby remained in the lungs (Fig. 1C). The molecular mechanisms responsible for the enhanced adhesion of T. gondii-infected leukocytes to the vascular endothelium are not known in detail. However, it is known that even a single T. gondii parasite within a human monocyte can impair integrin clustering on the surface of the monocyte and alter the dynamics of monocyte adhesion to the vascular endothelium [13].

TRANSFER OF TACHYZOITES FROM LEUKOCYTES TO PARENCHYMAL TISSUES

There are two hypotheses as to how tachyzoites are transferred from infected leukocytes arriving at the target organ to the parenchymal cells of the target organ [6]. The first hypothesis is that leukocytes carrying internal tachyzoites emerge from the blood vessels by extravascular migration and transport the tachyzoites to parenchymal tissues (Trojan Horse Hypothesis). The other is that tachyzoites egress from the leukocytes then cross the vascular endothelium on their own (Hitchhiker’s Hypothesis) (Fig. 1D). When we observed infected leukocytes entering the lungs, it was evident that the tachyzoites egressed out from the leukocytes within hours of their influx to the lungs. In other words, it was clear that the Hitchhiker’s Hypothesis was correct, at least in the lungs [1]. In this study, we also revealed that the adherence of infected leukocytes to the endothelium of the lungs triggered tachyzoite egress (Fig. 1E) [1]. Konradt et al. reported that tachyzoites reaching the brain via the bloodstream invaded the interior of vascular endothelial cells and proliferated there [11]. In the brain, the “Hitchhiker’s Hypothesis” is more likely to be correct than the “Trojan Horse Hypothesis”. T. gondii tachyzoites invade various organs, including the lungs and brain. In the Hitchhiker’s Hypothesis, the intracellular parasite must egress out from the infected leukocytes at the same time that infected leukocytes adhere to the microvascular endothelium of the target organ (Fig. 1D). Although the molecular mechanism of regulation for the parasite egression is unknown, we found that anti-CD162 antibodies prevented parasite egression. It is possible that CD162 molecules on host cells are involved in this regulated egression [1]. It is also known that crosstalk between the protein kinase A catalyst and cGMP-dependent protein kinase G, both of which are encoded by T. gondii, govern T. gondii egress [8]. The adhesion of parasite-infected leukocytes to the vascular endothelium may cause an abrupt change in the activity of these enzymes, resulting in egress of the parasite inside the leukocytes.

It is still unclear whether the “Hitchhiker’s Hypothesis” is always correct for all organ invasions or whether the “Trojan Horse Hypothesis” may be correct for some organs. This is an issue for future research; however, we believe that it can be solved by applying the methods we have used to other organs. The research methods presented herein can also be applied to other intracellular parasitic pathogens. Studies using in vitro blood-brain barrier models have favored the “Trojan Horse Hypothesis” for the mechanism by which the fungal pathogen Cryptococcus neoformans crosses the blood-brain barrier [19]. It is not yet known whether C. neoformans behaves in the same manner in vivo as predicted in the vitro model, but an approach similar to that applied to study T. gondii in the host body may be useful.

CONCLUSION

Increasing the transparency of the organs of experimentally infected animals by the application of fluorescently-labeled parasites was very effective in helping us elucidate the behavior of parasites in the host body. Using this technique, we clarified how T. gondii disseminates throughout the host body from the site of entry via the circulatory system and transfers from the circulatory system to the parenchyma of the target organs.

CONFLICT OF INTEREST

The authors declare no conflict interest.