When we left for the brain in a DC shuttle - Toxoplasma gondii

If you know me well, you would know my near-fanatical obsession with the awe-inspiringly beautiful Aurora Borealis. They are especially delightful on cold wintry nights on the icy, wind-swept prairie. And out in the darkness, when one sits, waiting for the beautiful ribbons of light to appear, one may observe the bright, distant balls of gas that are scattered across the sky in a milky band, and sometimes the man-made satellites that look like fast moving stars, seemingly weave in and out of the constellations in the background. If you asked me, I would tell you that astrophysics, sans the formulae, is certainly a field meant for hobby reading. Recently, I enjoyed a documentary series, produced by Discovery Communications, titled "When we left Earth : The NASA Missions", which featured the Mercury, Gemini, Apollo Missions, and NASA's golden age, the Space Shuttle era. The progress in the technology and the ability of humans to go beyond the physical boundaries imposed by the planet and its forces are quite remarkable, and quite interesting to follow, all the more if you were old enough to remember the landing on the moon, [which I am not, but that is not a major deterrent].

Just as the space shuttles were designed to carry payloads and people to the low Earth orbit, the zoonotic apicomplexan, Toxoplasma gondii , uses the dendritic cells of the host to move around uninhabitable spaces and cross barriers that it normally cannot by itself cross. Most comparisons call for the dendritic cells to play the role of a Trojan horse, but the space shuttle analogy works just as well.

Here is a brief overview of the life cycle before we scrutinize Toxoplasma's insane abilities to flag down, hitch rides and hijack shuttles. When the infective bradyzoites (in the cyst) are ingested by the definitive host (domestic cat and other Felids), the sexual cycle ensues in the intestines and oocysts are shed in the feces; quite similar to other Coccidians. However, the asexual cycle in the intermediate hosts is what sets Toxoplasma apart. In humans, sheep, mice and every other imaginable host, infective oocysts differentiate into tachyzoites which invade the intestines, multiply and later migrate in the body to form cysts containing bradyzoites. Favorite spots for cyst formation include the brain, the placenta (resulting in abortion(s)) and the eyes; every one of these organs are "immuno-previleged", that is, they are places where innate immuno-surveillance is lower than in the other organs of the body. Before cysts can be formed, Toxoplasma gondii  has to get to these places.The migration of a stage called the tachyzoite is the theme of this post.


Getting to know your shuttle
The Dendritic cells are an enigmatic group of leukocytes, first discovered by Steinman R. in 1973, (Steinman was awarded the 2011 Nobel Prize in Physiology/Medicine, only three days after his death. You know of course, that the Prize is usually not awarded posthumously and this was a rare exception made in good faith by the Nobel Prize Committee, whose members were entirely unaware of the death of the laurate) , that have an absolutely incredible range of activities to perform. These bone marrow derived cells, migrate to their temporary homes and keep themselves entertained by sampling their environment, taking up and processing antigens. They are the great controllers of the adaptive immune system, the gatekeepers, if you will, and live throughout the body, at all possible portals of pathogen entry (the skin, the intestines etc), and the secondary lymph organs. They play key roles in immunological tolerance. Once they encounter an antigen that is deemed dangerous, based on its molecular phenotypic profile, or in simple terms the pattern of its dress, they take it up, process it (read "beat it to a pulp") and later migrate with it to lymph nodes where they "present" the processed antigen (on silver platters called MHC molecules) to T cells that are fit to receive such presents (viz., antigen specific). All the while, the DCs secrete chemokines, through which other cells are activated and the immune response spirals into a cell mediated or humoral response as necessary.

DCs exist as distinct subtypes. The jury is out on whether these subtypes are specialized lineages or just functionally plastic cells. The cellular marker of a mature DC in mouse is the CD11c molecule. Interdigitating DCs carry CD205 as well. CD11b is another marker of interest. In mice spleen, CD4-CD8+, CD4+CD8- and CD4-CD8- DC populations exist. Thymic DCs in mice are predominantly CD4-CD8+CD205+CD11b-. Lymph node DCs are predominantly CD4-CD8-CD205-CD11b+ cells. Langerhans DCs dominate the skin draining LNs, and are CD4-CD8-CD205+CD11b+.

Toxoplasma gondii, the perfect parasite, makes use of the migratory activity of this mysterious cell type. In the intermediate host (the animals on the left side on the picture above; that is, mice, humans and other mammals), the parasite enters the body through the mouth when the infective oocyst stage is accidentally consumed. Oocysts that are shed by a household cat is usually the reason that humans come in contact with the parasite. Toxoplasma oocysts unmask themselves to show their virulent side, becoming fast replicating tachyzoites, by invading the intestinal cells and replicating. The DC in the vicinity leap into action. But, even as they lash out, Toxoplasma tachyzoites invade them and causes them to become hypermotile, even in the absence of chemotactic stimuli from other predilection sites. The precise mechanism by which it uses the DCs as its own personal mode of transportation is only slightly known. Within 2 - 3 hrs the parasite rides its shuttle to the local lymph node, each content to bring the other to the LN.

Leaping to action
The DC response at the first hint of the tachyzoites is the production of IL12, triggered through TLR 11 recognition of a profilin-like molecule of the parasite and CCR5 mediated recognition of cyclophilin 18. Other TLRs that are activated include TLR2 and TLR4, both of which are activated by Toxoplasma's glycosylphosphotidylinositol anchors. Once the TLRs are activated, the response is rapid. IL12 activates NK cells, which secrete the first doses of interferon gamma. By this time, the DCs have migrated to the lymph node and present the Profilin-like molecule on MHC Class II molecules. DCs continue to secrete IL12. Antigen specific CD4+ cells are activated by the cytokine mileu and the presented antigen. They begin to pour out IFN gamma in a perfect example of a positive feedback loop, driving their own differentiation down a Th1 road. And you probably know that Th1 responses are primarily cell mediated. Also, DCs are capable of direct antigen presentation on MHC Class I molecules to CD8+ cells, activating them. Actively dividing tachyzoites are now cleared by the CD4+ and CD8+ cells that have migrated to the infection sites in the intestines and the acute phase of the infection ends. The immune system seems to have done its job.

However, the tachyzoites that infected the DCs that have now reached the local lymph node are alive and well. Once a DC reaches the lymph node, it does not ordinarily leave very easily under normal circumstances. Toxoplasma infected DCs though exhibit a bizarrely hypermotile phenotype that allows them to be steered as Toxoplasma pleases. The CD 11c on the surface is down regulated and the CD 11b is upregulated. The infected cells turn from CD11c+CD11b+/- to CD11c-CD11b+.

According to a study by Lambert et al. (2009), hypermotility of the DCs also depends on the genotype of Toxoplasma, with Type II strains showing "stronger intracellular associations with CD11c+ DCs". Types II and III were better at causing DC movement than Type I, although the latter seemed to have better motility on its own, and was able to cross physiological barriers than the former two.

The destination
In an elegant review article, Galea et al. (2006) clarify the 'immune privileged' status experienced by the brain, which is where Toxoplasma controlled DCs are headed. They reiterate that "immune privilege is relative and not absolute", that is, whilst the brain in its normal condition does not host DCs and other antigen presenting cells, an inflammatory response is an altogether different story. One possible reason for this according to other neuroscientists including Coisne et al. 2006 is the "Differential expression of selectins" and other adhesins like ICAM-1 and VCAM-1.

Barragan et al. in 2005 have shown that when Toxoplasma transmigrates, that is,  moves across the blood brain barrier without disrupting it, the layers of intervening cells maintain their structure and integrity. And because this was studied in an electrical organ like the brain, they also measured the Transcellular electrical resistance, which they found to be maintained at a steady state during said parasite-DC migration. Using ICAM-1 antibodies, they discovered that ICAM-1 on the host cell surface was involved and that it interacted with a set of micronemal proteins on Toxoplasma called MIC2.

In a more recent study, Lachenmaier et al. (2011) pinned the exact mechanism of neuroinvasion by stating that CD11b+ cells were the main cell population that were used. They further claimed that both type I and II strains were equally capable of infecting CD11b+/CD11c+ cells and migrating across the blood brain barrier. Transmigration also seemed to cause "a marked change in the transcriptome of the brain endothelial cells", which basically caused the upregulation of adhesins, integrins and selectins, including ICAM-1, E-selectin, P-selectin and TLR4. Other cytokines whose transcriptions were upregulated include IL6, MCP-1 and the cytokines CCL2, CCL7, CXCL1, CXCL2 and CX3CL1. Since the entry is essentially paracellular, and because the parasite must continue to evade the immune system, they found that certain molecules were downregulated. These included claudin 8, that belongs to the claudin group of proteins that are involved with sealing of the tight junctions. Downregulation of claudin 8 causes loosening of the junctions. Interferon gamma production was downregulated as well, by the downregulation of the transcription factor STAT4. The authors suggest that preliminary evidence exists to support the hypothesis that extracellular tachyzoites are not as capable of crossing the BBB as ones that are infecting leucocytes.

In conclusion, Toxoplasma gondii seems to be a parasite that is more than capable of controlling the host immune response in all the ways necessary to achieve its purposes of entering the brain. Once in the brain, the parasite forms cysts that contain a slowly replicating form (bradyzoites). This causes a latent form of the disease, that in humans occasionally manifests as Toxoplasmic encephalitis or TE. TE patients exhibit neurological symptoms that may include schizophrenia. The exact prognosis of infection depends on various controllable and non-controllable factors that are wonderfully reviewed by Carruthers and Suzuki, 2007. Although it is predominantly an animal parasite, the zoonotic nature of Toxoplasma surely compels our attention.

References in no particular order :
Sanecka A, Frickel EM. Use and abuse of dendritic cells by Toxoplasma gondii. Virulence. 2012 Nov 15;3(7):678-89.Review.

Feustel SM, Meissner M, Liesenfeld O. Toxoplasma gondii and the blood-brain barrier. Virulence. 2012 Mar-Apr;3(2):182-92.

Lachenmaier SM, Deli MA, Meissner M, Liesenfeld O. Intracellular transport of Toxoplasma gondii through the blood-brain barrier. J Neuroimmunol. 2011 Mar;232(1-2):119-30.

Carruthers VB, Suzuki Y. Effects of Toxoplasma gondii infection on the brain. Schizophr Bull. 2007 May;33(3):745-51. Epub 2007 Feb 23. Review.

Galea I, Bechmann I, Perry VH. What is immune privilege (not)? Trends Immunol. 2007 Jan;28(1):12-8.

Denkers EY, Butcher BA, Del Rio L, Bennouna S. Neutrophils, dendritic cells and Toxoplasma. Int J Parasitol. 2004 Mar 9;34(3):411-21. Review.

Barragan A, Brossier F, Sibley LD. Transepithelial migration of Toxoplasma gondii involves an interaction of intercellular adhesion molecule 1 (ICAM-1) with the parasite adhesin MIC2. Cell Microbiol. 2005 Apr;7(4):561-8.

Coisne C, Faveeuw C, Delplace Y, Dehouck L, Miller F, Cecchelli R, Dehouck B. Differential expression of selectins by mouse brain capillary endothelial cells in vitro in response to distinct inflammatory stimuli. Neurosci Lett. 2006 Jan 16;392(3):216-20. Epub 2005 Oct 7.

Lambert H, Vutova PP, Adams WC, Loré K, Barragan A. The Toxoplasma gondii-shuttling function of dendritic cells is linked to the parasite genotype. Infect Immun. 2009 Apr;77(4):1679-88.

"Toxoplasmosis life cycle en" by LadyofHats. Licensed under Public domain via Wikimedia Commons - http://commons.wikimedia.org/wiki/File:Toxoplasmosis_life_cycle_en.svg#mediaviewer/File:Toxoplasmosis_life_cycle_en.svg