Toxoplasma gondii effectors are master regulators of the inflammatory response

Abstract

Toxoplasma is a highly successful parasite that establishes a life-long chronic infection. To do this, it must carefully regulate immune activation and host cell effector mechanisms. Here we review the latest developments in our understanding of how Toxoplasma counteracts the immune response of the host, and in some cases provokes it, through the use of specific parasite effector proteins. An emerging theme from these discoveries is that Toxoplasma effectors are master regulators of the pro-inflammatory response, which elicits many of the toxoplasmacidal mechanisms of the host. We speculate that combinations of these effectors present in certain Toxoplasma strains work to maintain an optimal parasite burden in different hosts to ensure parasite transmission.

Figure 1. Host cell responses that can be modulated by Toxoplasma gondii

(1) Toll-like receptors (TLRs) are activated upon recognition of pathogen associated molecular patterns (PAMPs). The main TLR ligand identified in T. gondii is a parasite profilin-like protein (TgPRF) that can bind to and activate TLR11 [59, 92]. Toxoplasma is also armed with molecules of glycosylphosphatidylinositol anchors (GPI) and glycoinositolphospholipids (GIPLs) that can be recognized by TLR2 and TLR4 [93]. (2) TLR engagement triggers MyD88-dependent signaling pathways that culminate with the activation of NF-kB. However, T. gondii strains that express the active form of the dense granule protein GRA15 are able to directly activate NF-κB through a MyD88-independent mechanism. (3) NF-κB activation leads to transcription of a series of pro-inflammatory genes, including genes for IL-1β, IL-12, IL-18, induced nitric oxide synthase (iNOS) and some NOD-like receptors (NLRs). Nevertheless, parasite ROP16 is able to suppress the IL-12 response of infected macrophages stimulated with the TLR agonists [41] and to inhibit NF-κB transcriptional activity [42], possibly due to its ability to phosphorylate and activate STAT3/6 [41], which dampens TLR-induced cytokine production. Parasite induced MAPK signaling pathways also modulate IL-12 production [94], and there is evidence that T. gondii ROP38 may regulate MAPK function [7]. (4) Binding of ATP to the purinergic receptor P2X₇ and the subsequent efflux of intracellular K⁺ leads to activation of the inflammasome [52, 53]. Although it is not known if Toxoplasma infection affects P2X₇R function, the parasite secretes nucleoside triphosphate hydrolases (NTPases) that could possibly control extracellular levels of ATP. (5) Inflammasome stimulation activates caspase-1, which cleaves the proforms of IL-1β and IL-18 generating bioactive cytokines. Both IL-1β and IL-18 receptors activate NF-κB and MAPK signaling and subsequent pro-inflammatory cytokine production. Toxoplasma is known to induce IL-1β and IL-18 secretion, both of which serve to amplify IFNγ production by NK cells [80, 81]. It remains to be elucidated if the parasite can directly activate the inflammasome or modulate caspase-1 activity. (6) IFNγ binding to its receptor triggers the JAK/STAT pathway, leading to phosphorylation of STAT1. Phosphorylated STAT1 then dimerizes and translocates to the nucleus, leading to transcription of interferon-stimulated genes, including the transcription factor IRF1, class II MHC and interferon regulated GTPases (IRGs). Yet, Toxoplasma infected cells display a marked inhibition of STAT1 dependent transcription [60], and parasite secreted kinase ROP18 can phosphorylate and inactivate IRGs (7), preventing their accumulation on the parasitophorous vacuole membrane and protecting the parasite from IRG-dependent intracellular killing [28, 29]. Abbreviations: IRF1, interferon regulatory factor 1; JAK, Janus kinases; MAPK, mitogen-activated protein kinase; STAT, signal transducer and activator of transcription; ROS, reactive oxygen species.

Figure 2. Overview of how Toxoplasma strains modulate host immune pathways

Modulation of host cell signaling pathways requires the secretion of numerous parasite proteins from specialized secretory organelles called dense granules and rhoptries. At early time points, infection with type I parasites does not activate pro-inflammatory responses. The type I (RH strain) allele of GRA15 results in a truncated and non-functional protein, allowing a ‘silent’ infection without activation of NF-κB [42]. On the other hand, ROP16_I induces sustained activation of STAT3 and STAT6, dampening the production of IL-12, IL-1β and IL-6 [41]. Together with the ability to reduce pro-inflammatory cytokine production, type I parasites express ROP5 alleles associated with high virulence [26, 37], and ROP18_I phosphorylates IRGs blocking their recruitment to the PV, which is required for parasite destruction, permitting unrestricted parasite growth [28, 29]. Conserved parasite proteins secreted by infected cells, profilin and cyclophylin-18, are recognized by DCs via TLR11 and CCR5 respectively, leading to late NF-κB activation and production of IL-12, which in turn activates NK and T cells to secrete IFNγ [59, 95]. However, type I parasites also prevent activation of DCs [96], and by the time that the pro-inflammatory response kicks in, host survival is already compromised due to uncontrolled parasite burden. Type II parasites are very effective in activating an early response. These parasites express the active form of GRA15, which activates NF-κB in the infected cells [42], and a less functional form of ROP16, which leads to a transitory activation of STAT3/6 [41]. As a consequence there is a massive production of pro-inflammatory cytokines early after infection. The environment induced by the parasite modulates activation of several T cell subtypes, mainly directing the response towards a Th1 type [97]. Aspects of the Th17 response to Toxoplasma seem to have opposite effects on host survival, mainly an IL-23 driven IL-22 response by CD4 T cells has a negative effect [98], while signaling through the IL-17 receptor can have a beneficial effect by lowering parasite burden [99]. Intracellular parasite growth is controlled due to expression of an avirulent form of ROP18, which does not block the recruitment of IRGs to the PV [28, 29], and type II parasites also express ROP5 alleles associated with low virulence [26, 37], but susceptible animals die of severe ileitis [69]. Like type I, type III secreted GRA15 and ROP16 do not activate NF-κB and induce a sustained activation of STAT3/6 respectively, limiting the initial production of pro-inflammatory cytokines [41, 42]. Nevertheless, these parasites express an inactive ROP18, being unable to avoid intracellular killing mediated by IRGs [28, 29]. In this case, late production of IL-12 by DCs triggers a Th1-type response that is sufficient to control parasite burden and induce cyst formation, leading to a chronic infection. CCR5, C-C chemokine receptor type 5; DCs; dendritic cells; GRA, dense granule protein; IRG, interferon-regulated GTPase; NK, natural killer cells; NO, nitric oxide; PV, parasitophorous vacuole; ROP, rhoptry protein; STAT, signal transducer and activator of transcription; ROS, reactive oxygen species, TLR11, Toll-like receptor 11.