The ROP16III-dependent early immune response determines the subacute CNS immune response and type III Toxoplasma gondii survival

Abstract

Toxoplasma gondii is an intracellular parasite that persistently infects the CNS and that has genetically distinct strains which provoke different acute immune responses. How differences in the acute immune response affect the CNS immune response is unknown. To address this question, we used two persistent Toxoplasma strains (type II and type III) and examined the CNS immune response at 21 days post infection (dpi). Contrary to acute infection studies, type III-infected mice had higher numbers of total CNS T cells and macrophages/microglia but fewer alternatively activated macrophages (M2s) and regulatory T cells (Tregs) than type II-infected mice. By profiling splenocytes at 5, 10, and 21 dpi, we determined that at 5 dpi type III-infected mice had more M2s while type II-infected mice had more pro-inflammatory macrophages and that these responses flipped over time. To test how these early differences influence the CNS immune response, we engineered the type III strain to lack ROP16 (IIIΔrop16), the polymorphic effector protein that drives the early type III-associated M2 response. IIIΔrop16-infected mice showed a type II-like neuroinflammatory response with fewer infiltrating T cells and macrophages/microglia and more M2s and an unexpectedly low CNS parasite burden. At 5 dpi, IIIΔrop16-infected mice showed a mixed inflammatory response with more pro-inflammatory macrophages, M2s, T effector cells, and Tregs, and decreased rates of infection of peritoneal exudative cells (PECs). These data suggested that type III parasites need the early ROP16-associated M2 response to avoid clearance, possibly by the Immunity-Related GTPases (IRGs), which are IFN-γ- dependent proteins essential for murine defenses against Toxoplasma. To test this possibility, we infected IRG-deficient mice and found that IIIΔrop16 parasites now maintained parental levels of PECs infection. Collectively, these studies suggest that, for the type III strain, rop16III plays a key role in parasite persistence and influences the subacute CNS immune response.

The Toxoplasma gondii virulence factor ROP16 acts in cis and trans, and suppresses T cell responses

“…Recent work has shown that all strains of T. gondii use the effector protein TgIST to block STAT1 signaling and thus inhibit IFN-γ–associated antimicrobial mechanisms after day 5 of infection (Gay et al, 2016; Olias et al, 2016). Interestingly, the ability to use the CEP strain shows that the loss of ROP16 results in a marked reduction in parasite levels as early as 4–5 dpi, which is even more significant by 10 dpi and has been extended to the chronic phase of infection (Tuladhar et al, 2019). Here, transcriptional profiling of macrophages infected and injected by CEP in vivo showed that suppression of IFN-γ–dependent innate immune mechanisms was only observed in infected cells, consistent with the role of TgIST as a late effector protein.…”

“…Transgenic Toxoplasma -Cre strains of T. gondii (Pru-Cre-tdTomato) were generated as previously described (Koshy et al, 2010). The engineering of the CEPΔROP16III strain is fully described in Tuladhar et al (2019). In brief, CEPΔhpt was cotransfected with four plasmids: two engineered with guide RNAs to upstream or downstream 21-mers for the rop16 locus, the pTKO plasmid engineered to contain 500-bp ROP16 homology regions surrounding the hypoxanthine phosphoribosyltransferase hxgprt cassette and a toxofilin-Cre cassette, and one plasmid containing the coding sequence for tdTomato under the GRA2 promoter.…”

Transcriptional profiling reveals T cells cluster around neurons injected withToxoplasma gondiiproteins

“…5C, S3)-is intriguing. These data suggest that type II and type III strains may have different rates of conversion from tachyzoites to bradyzoites in vivo, with the type III strain trailing behind the type II strain at the D21 timepoint, despite the fact that type III strains are very capable of forming cysts in vivo 23,32,61 . We recently described how the host immune response at the same time point differs between these strains 32 , leading to a proverbial chicken-or-the-egg question.…”

“…Finally, to enable our ability to identify both universal and strainspecific neuron manipulations, we utilized mice infected with either of the canonical, persistent T. gondii strains. These strains-belonging to either the type II and type III lineages-are genetically distinct, express different polymorphs of some injected effector proteins 24,29,31,30,28,25,27 and are known to drive distinct CNS inflammatory responses 32 . Thus, with this combination of tools, we sought to define T. gondii's strain-specific and universal effects on neurons during an in vivo infection.…”

Transcriptional Profiling Suggests T Cells Cluster around Neurons Injected with Toxoplasma gondii Proteins

“…Yet, very little is known about the neuron-T. gondii interaction, especially in vivo. What we do know about host cell-T. gondii interactions-that T. gondii secretes many effector proteins into host cells prior to and after full host cell invasion and that these effector proteins can be polymorphic between T. gondii strains, leading to strain-specific host cell manipulations-comes primarily from in vitro studies in fibroblasts and immune cells (3,(24)(25)(26)(27)(28)(29)(30)(31)(32).…”