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Quantitative intravital imaging of Plasmodium falciparum sporozoites: A novel platform to test malaria intervention strategies

Christine S. Hopp, Sachie Kanatani, Nathan K. Archer, Robert J. Miller, Haiyun Liu, Kevin Chiou, Lloyd S. Miller, Photini Sinnis

Preprint posted on July 30, 2019 https://www.biorxiv.org/content/10.1101/716878v1

One for all: IVM shows comparable dynamics of human and rodent Plasmodium sporozoites in murine skin, offering a promising tool to investigate anti-malarial intervention targeting sporozoites at the point of inoculation.

Selected by Mariana De Niz

Background

Malaria infection begins with the injection of Plasmodium sporozoites into the host’s skin during a female Anopheles mosquito bite. Various studies have shown that sporozoite motility is essential for Plasmodium exit from the dermis after inoculation, invasion of the vasculature, and ultimately, infectivity. Multiple studies on parasite motility in vivo have benefited from intravital microscopy (IVM) which allows qualitative and quantitative assessment of host-pathogen interactions at sub-cellular resolution in situ within the living host (1-5). A consistent conclusion from current studies, is that the skin represents a site where Plasmodium sporozoites are vulnerable to antibody recognition.

P. falciparum is responsible for most deaths among humans, and causative of severe complications. However, most of our in vivo knowledge on sporozoite gliding motility for infectivity has arisen from rodent models, and functional in vivo studies on the human-infective P. falciparum sporozoites have never been performed. A common discussion in Plasmodium research focuses on the extent to which observed phenomena such as pathology, vaccine efficacy, and host-parasite interactions transcend species-specific findings; i.e. whether findings in rodent models are relevant to humans. In their work, Hopp et al. (6) compare the behaviour of human and rodent Plasmodium sporozoites in vivo, in both murine and xenografted human skin.

 

Key findings and developments

 Overall key findings

  • The work by Hopp et al. (6) presents the first characterization of P. falciparum sporozoites in the skin, comparing their motility to two rodent malaria species, P. berghei and P. yoelii, by quantitative IVM (Figure 1).
  • They found that as opposed to blood and liver stages, which are species-specific, P. falciparum sporozoites move with similar speed, displacement and duration, and enter blood vessels in similar numbers as rodent parasites.
  • This finding is important as it shows that interventions targeting P. falciparum sporozoite migration can be tested in the murine dermis.
  • The authors also developed methods for automated detection and tracking of sporozoites in IVM videos. This will allow high throughput testing and analysis of anti-malarial interventions targeting sporozoites.
Figure 1. Maximum intensity projection of P. berghei, P. yoelii and P. falciparum sporozoite trajectories (green) over time, relative to blood vessels (magenta), 10 minutes after inoculation in the skin (Adapted from ref. 6).

 

 

Automated sporozoite detection and tracking

  • To streamline quantification of sporozoite motility, the authors developed an automated method to detect and track sporozoites using FIJI for background subtraction and thresholding of raw images, and ICY for detection and tracking.
  • The automated method generated data comparable to the data obtained with manual sporozoite tracking previously used (2).

 

Biological findings in murine skin

  • Using the automated detection and tracking method, P. berghei, P. yoelii and P. falciparum motility in the dermis of mice was compared.
  • For all 3 Plasmodium species, the percentage of the sporozoite population moving large distances (75-200 µm) was maximal at 5-20 min. However, while almost 10% of P. berghei parasites move this far at 10 minutes post-inoculation, only around 3% of P. falciparum sporozoites do.
  • While for P. berghei and P. falciparum, high-displacing sporozoites are no longer observed at 60 min post-inoculation, P. yoelii sporozoites were still moving larger distances even 120 min after inoculation.
  • At 5-10 minutes after inoculation, sporozoites of all 3 Plasmodium species move at approximately 1.5 µm/s. Compared to P. berghei, which begins to slow down significantly by 60 min post-inoculation, P. yoelii and P. falciparum gliding speeds stay more constant over time.
  • P. berghei, P. yoelii and P. falciparum frequently circle around CD31+ vessels, likely to optimize contact, and there were no statistically significant differences in the rate of blood or lymphatic vessel entry among the 3 Plasmodium species.

 

Biological findings in humanized rodent models

  • The authors developed an in vivo humanized mouse model of P. falciparum skin infection to investigate sporozoite motility, by grafting human skin onto NSG mice. The grafted human skin retains the human vasculature, and blood supply to the graft is restored through spontaneous anastomosis of murine and human vessels.
  • The mean displacement of the P. falciparum sporozoite population in the human skin xenograft was lower than in murine dermis, at all time points after inoculation. Furthermore, sporozoites moved with significantly reduced speed at most time points in human skin compared with mouse skin. A hypothesis for these observations was the presence of blood vessel anastomoses, which could affect track linearity and thereby impact both sporozoite displacement and speed.
  • P. falciparum sporozoites move in more constrained circular paths in the grafted human skin. Additionally, comparison between measurements in live xenografted mice, with grafts analysed ex vivo showed that sporozoite tracks ex vivo are significantly less confined than those in the grafted human skin (with blood flow), and similar to the observations in mouse skin.

 

What I like about this paper

I like this pre-print because it is the first study comparing the rodent and human Plasmodium species in vivo. Furthermore, the Sinnis lab has developed important tools for intravital microscopy, and here they contribute another piece of the toolbox, useful for automated high throughput analysis. Also, I like in this and previous work of their lab, that imaging is used for very detailed and thorough observations of the parasite in its environment. This careful level of observation and analysis has led their lab to important and relevant findings in the field.

Their contribution also is important given some hesitation in the malaria field, on what the extent of the usefulness of rodent models to study Plasmodium is, and ultimately the relevance of in vivo studies for the development of anti-malarial interventions.

 

Open questions

  1. As you mention in the discussion and introduction, unlike blood and liver stages which are strictly species-specific, the skin stage does not seem to be so. From an evolutionary perspective, why do you think this is so? Is it possible that movement in the skin is to a large extent, substrate-related, and that sporozoites of all Plasmodium species would move in the same way, should a substrate with the same overall characteristics as the skin be present? In terms of sensing, are there indications that different Plasmodium species sense receptors in the vascular endothelium or chemotactic factors differently?
  2. In your work, you also analysed the differences between the two rodent Plasmodium species, P. yoelii and P. berghei. One of the findings you made, which corroborates previous observations, is that P. yoelii sporozoites move more persistently, with higher speed and larger displacements at later time points than P. berghei. Why do they behave differently? Since one of the comparisons is the relevance of the host, would the two murine models show more similar dynamics in their original hosts (i.e. thicket rats), than in inbred mouse strains? How do the murine strains behave in the xenograft?
  3. Previous studies have described sporozoite dynamics being different in the ear pinna than in skin flaps at other body sites, due to different vascular density and temperature, among other factors.  Do you think the dynamics you observed for the three species, are similar across different skin locations in the mouse?
  4. Some groups have used organs on chips, or organoids to study host-pathogen interactions. For Plasmodium sporozoites, what advantages do you see in an IVM-based method, over organoids or organs on chips?
  5. Previous work showed that sporozoites are able to invade cells in the skin, and develop within them. Did P. falciparum do this either in the xenografted skin or in the mouse skin?

References

  1. Vandenberg JP, Frevert U. Intravital microscopy demonsztating antibody-mediated immobilization of Plasmodium berghei sporozoites injected into skin by mosquitoes. Int J Parasitol (2004) 34:991-996. doi:10.1016/j.ijpara.2004.05.005.
  2. Hopp CS, Chiou K, Ragheb DR, Salman A, Khan SM, Liu AJ, Sinnis P., Longtitudinal analysis of Plasmodium sporozoite motility in the dermis reveals component of blood vessel recognition. Elife (2015) 4: doi: 10.7554/eLife.07789.
  3. Amino R, Thiberge S, Martin B, Celli S, Shorte S, Frischknecht F, Menard R. Quantitative imaging of Plasmodium transmission from mosquito to mammal. Nat Med. (2006) 12:220-224. doi:10.1038/nm1350.
  4. Hellmann JK, Münter S, Kudryashev M, Schulz S, Heiss K, Müller AK, Mattuschewski K, Spatz JP, Schwarz US, Frischknecht F. Environmental constrains guide migration of malaria parasites during transmission. PLoS Pathog. (2011) 7:e1002080. doi:10.1371/journal.ppat.1002080.
  5. Flores-Garcia Y, Nasir G, Hopp CS, Munoz C, Balaban AE, Zavala F, Sinnis P. Antibody-mediated protection against Plasmodium sporozoites begins at the dermal inoculation site. mBio (2018) 9(6), pii: e02194-18. doi: 10.1128/mBio.02194-18.
  6. Christine S. Hopp, Sachie Kanatani, Nathan K. Archer, Robert J. Miller, Haiyun Liu, Kevin Chiou, Lloyd S. Miller, Photini Sinnis. Quantitative intravital imaging of Plasmodium falciparum sporozoites: a novel platform to test malaria intervention strategies. bioRxiv, (2019), doi: 10.1101/716878.

Acknowledgements

I am very grateful to Photini Sinnis and Christine Hopp for their time and willingness to engage in this preLight highlight, and the open and exciting discussion regarding their work.

 

Posted on: 28th August 2019

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  • Author's response

    Photini Sinnis and Christine Hopp shared

    Open questions

    1. As you mention in the discussion and introduction, unlike blood and liver stages which are strictly species-specific, the skin stage does not seem to be so. From an evolutionary perspective, why do you think this is so? Is it possible that movement in the skin is to a large extent, substrate-related, and that sporozoites of all Plasmodium species would move in the same way, should a substrate with the same overall characteristics as the skin be present? In terms of sensing, are there indications that different Plasmodium species sense receptors in the vascular endothelium or chemotactic factors differently?

     The initial phase of malaria infection in the dermal inoculation site is the only time the parasite is extracellular for an extended period and as you suggest, it is likely that the signals the sporozoite receives as it moves in the mammalian host, are similar among different Plasmodium species. It may be responding to temperature and/or pH changes, and as has been previously shown in vitro, sporozoites are likely also responding to the presence of a substrate (1) and possibly albumin (2). Though there are likely differences in the composition of extracellular matrix between mouse and human, critical components such as collagen type may be the same. Similar to gametocyte activation in the mosquito host, sporozoites likely respond to some changes that are general to the mammalian host though the precise nature of these factors remains unknown.  Regarding blood vessel recognition, though it is clear from our previous work (3) that sporozoites recognize blood vessels, precisely what they are sensing remains an unanswered but important question. Our somewhat surprising finding that P. falciparum sporozoites enter mouse blood vessels with equal efficiency to their rodent malaria counterparts, suggests these factors are common to mammalian hosts.

     

    1. In your work, you also analysed the differences between the two rodent Plasmodium species, P. yoelii and P. berghei. One of the findings you made, which corroborates previous observations, is that P. yoelii sporozoites move more persistently, with higher speed and larger displacements at later time points than P. berghei. Why do they behave differently? Since one of the comparisons is the relevance of the host, would the two murine models show more similar dynamics in their original hosts (i.e. thicket rats), than in inbred mouse strains? How do the murine strains behave in the xenograft?

    As you point out, the laboratory mouse is not the natural host for either rodent species and we do not know how they would behave in their natural host, the thicket rat. This would be very interesting to determine and may show that differences between the rodent species disappear in their natural host. However, it is also possible that the two rodent species have different strategies, i.e. that P. berghei moves faster for a shorter period of time whereas P. yoelii moves more slowly over a longer period of time. We have not investigated their behaviour in human skin xenografts but this would be an important future line of investigation and could be relevant to sorting out these questions.

     

    1. Previous studies have described sporozoite dynamics being different in the ear pinna than in skin flaps at other body sites, due to different vascular density and temperature, among other factors. Do you think the dynamics you observed for the three species, are similar across different skin locations in the mouse?

     There is one study from the Frischknecht group that has shown that P. berghei sporozoites move in more linear tracks in the tail compared to the ear (4). Temperature and vascular density are not significantly different in different skin sites, but it is possible that the collagen density varies, though we have no information on the composition of the skin extracellular matrix in different skin sites. We have previously quantified sporozoite motility in skin from the dorsal skin of the mouse and saw no differences from what we observed in the ear. Furthermore, in a study that is in preparation, we found no difference in infectivity of P. yoelii sporozoites when inoculated into the ear, back or tail of a mouse. Thus, while the types of motility we see may change in different skin beds, the overall ability of sporozoites to move and find blood vessels is likely similar across different locations.

     

    1. Some groups have used organs on chips, or organoids to study host-pathogen interactions. For Plasmodium sporozoites, what advantages do you see in an IVM-based method, over organoids or organs on chips?

     This is indeed an exciting area of investigation and for studies of liver invasion it could potentially be ground-breaking since the liver is such a difficult organ to access and observe in vivo. However, for the skin, which is readily accessible, we think that our intravital model is likely simpler and therefore don’t think that skin on a chip would be significantly more informative than what we have currently developed.

     

    1. Previous work showed that sporozoites are able to invade cells in the skin, and develop within them. Did P. falciparum do this either in the xenografted skin or in the mouse skin?

     Very interesting question. This would require us to process samples 5 to 7 days post inoculation and look to see whether there are developing exoerythrocytic stages (EEFs), which we did not do. This could be studied in future experiments. Importantly, only a very small proportion of the sporozoite inoculum develops into skin EEFs, with P. berghei it’s on the order of 1% and with P. yoelii it’s 0.5% (5-6). While these previous studies show that these skin EEFs cannot seed the blood to initiate a blood stage infection, it remains possible that they impact the host immune response to liver stage parasites. We do not know if these skin EEFs occur because the rodent parasites are not in their natural host or if they are a bonafide component of natural infection. Our human xenograft model could be used to determine if P. falciparum sporozoites develop into EEFs in the skin.

     

    References

    1. Pershchmann N, Hellmann JK, Frischknecht F, Spatz JP. Induction of malaria parasite migration by synthetically tunable microenvironments. Nano Lett. (2011), 11(10):4468-4474), doi:10.1021/nl202788r
    2. Vanderberg JP. Studies on the motility of Plasmodium sporozoites. Protozool., (1974) 21(4):527-537
    3. Hopp CS, Chiou K, Ragheb DR, Salman A, Khan SM, Liu AJ, Sinnis P., Longtitudinal analysis of Plasmodium sporozoite motility in the dermis reveals component of blood vessel recognition. Elife (2015) 4: doi: 10.7554/eLife.07789.
    4. Hellmann JK, Münter S, Kudryashev M, Schulz S, Heiss K, Müller AK, Mattuschewski K, Spatz JP, Schwarz US, Frischknecht F. Environmental constrains guide migration of malaria parasites during transmission. PLoS Pathog. (2011) 7:e1002080. doi:10.1371/journal.ppat.1002080.
    5. Voza T, Miller JL, Kappe SH, Sinnis P. Extrahepatic exoerythrocytic forms of rodent malaria parasites at the site of inoculation: clearance after immunization, susceptibility to primaquine, and contribution to blood stage infection, Immun. (2012), 80 (6): 2158-2164. doi: 10.1128/IAI00246-12.
    6. Gueirard P, Tavares J, Thiberge S, Bernex F, Ishino T, Milon G, Franke-Fayard B, Janse CJ, Menard R, Amino R. Development of the malaria parasite in the skin of the mammalian host, Natl. Acad. Sci. (2010), 107(43): 18640-18645. doi:10.1073/pnas.1009346107.

     

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