Toll-Like Receptors (TLR) play an essential role in sensing and responding to pathogens by the innate immune system [88
]. Signaling through TLR leads to induction of proinflammatory cytokines and chemokines, as well as anti-microbial molecules such as inducible nitric oxide synthase. To date, 11−13 TLR molecules have been identified in mammals. Most are expressed on the cell surface, but some are expressed in intracellular compartments (TLR3, 7, 8 and 9). One of the best characterized is TLR4, which, along with MD2, serves as a receptor for Gram-negative bacterial lipopolysaccharide (LPS). With the exception of TLR3, activation through these receptors requires recruitment of adaptor molecule MyD88, in turn resulting in recruitment of IL-1 receptor-associated kinases (IRAK) 1 and 4 (). These molecules form a complex with TNF receptor-associated factor (TRAF)6, resulting in interaction with Uva1 and Ubc13, leading in turn to ubiquitination of TRAF6. Then, ubiquitinated TRAF6 activates transforming growth factor-β-activated kinase (TAK)-1. Interacting with TAK1-binding proteins (TAB)-1 and -2, TAK1 serves as a mitogen-activated kinase (MAPK) kinase kinase, triggering the MAPK cascade (). The TAK1 molecule also activates the IκB kinase complex. This results in phosphorylation-dependent ubiquitination and degradation of IκBα, enabling nuclear translocation of NFκB.
Figure 2 The Toll-like receptor signaling cascade: Impact of Toxoplasma infection. In a generalized pathway, TLR binding to its ligand (step 1) results in recruitment of the MyD88 adaptor molecule (step 2). In turn, this mediates recruitment of IRAK1 and IRAK4, (more ...)
After invasion of macrophages, the Toxoplasma
Type 1 strain RH actively down-regulates a large panel of proinflammatory cytokines and chemokines that are normally induced by TLR signaling [50
]. We observed in particular that LPS-triggered IL-12p40 and TNF-α are suppressed in infected cells. While the parasite itself eventually initiates IL-12 synthesis, TLR4-triggered production of TNF-α remains potently suppressed. The ability of T. gondii
to block LPS triggered responses requires active invasion. Thus, heat inactivated tachyzoites do not display suppressive activity, and when parasite entry is prevented by cytochalasin D blockade of actin polymerization, suppressive activity is also lost [91
]. The suppressive activity of Toxoplasma
on LPS-induced TNF-α requires parasite survival within the host cell. This is because drug-induced tachyzoite inactivation after invasion restores the ability of cells to respond to TLR4 triggering [91
]. We also recently found that signaling through other TLR is blocked during infection [92
]. Importantly, this includes TLR3, an intracellular receptor for double-stranded RNA that, unlike other TLR, signals in a manner independent of MyD88 (). Suppression of TLR signaling does not appear to be restricted to RH strain tachyzoites because other Type 1, as well as Type 2, strain parasites also blocks LPS induction of TNF-α [92
Figure 3 T. gondii down-regulates MyD88-independent signaling mediated by poly I:C/TLR3. A, bone marrow-derived MyD88+/+ and MyD88−/− macrophages were infected, subjected to 2 hr poly I:C stimulation, then cells were harvested and RNA prepared (more ...)
The block in TLR signaling has also been reported to occur in bone marrow-derived dendritic cells [93
]. In this case, infected immature dendritic cells fail to mature in response to LPS triggering, and the cells were deficient in their ability to activate T cells. During in vivo infection, we also obtained evidence that Toxoplasma
blocks cytokine production in infected cells. Infected macrophages collected from the peritoneal cavity following i. p. parasite inoculation are suppressed in their ability to produce TNF-α, and infected dendritic cells in the spleen are defective in IL-12 production [14
]. Thus, suppression of TLR signaling by T. gondii
appears to be a general phenomenon that is not parasite strain restricted and that occurs in several cell types.
Deactivation of TLR signaling by Toxoplasma
may indicate the need for the parasite to avoid triggering these pathways by the parasite's own TLR ligands. Thus, the Toxoplasma
profilin molecule TGPRF activates TLR11 and parasite glycosylphosphoinositol (GPI) moieties associated with tachyzoite surface proteins possess the ability to activate TLR2 and TLR4 [5
]. Since both profilin and GPI synthesis are essential for survival [94
], the parasite may be under evolutionary pressure to block TLR signaling during intracellular infection. In this regard, the immunodominant CD4+
T cell response to TGPRF characterized by Yarovinsky and colleagues [96
] might result from recognition of this TLR11 ligand by noninfected antigen presenting cells. It is also possible that blocking TLR signaling is a means to prevent proinflammatory responses that would otherwise be triggered by exposure to gut flora now known to occur during oral T. gondii
]. Another possibility is that inability to respond to TLR ligands reflects a general nonresponsiveness of cells to proinflammatory signals no matter what the initiating stimulus, rather than being specific for TLR pathways.
There is evidence that T. gondii
inhibits TLR signaling through both NFκB and MAPK pathways. During early infection of macrophages, the parasite prevents accumulation of nuclear NFκB in response to LPS [50
]. This may be a consequence of failure to retain this transcription factor in the nucleus, rather than a block in nuclear import [52
]. The defect in NFκB nuclear accumulation is not permanent, because cells translocate this transcription factor in response to LPS when stimulation is performed 6 hr or more after infection [99
] (). Although we and others do not see RH-induced NFκB nuclear translocation, others have observed activation of this transcription factor during infection. There is also evidence that Type 2 T. gondii
strains themselves induce NFκB activation, and indeed, IL-12 production induced by the Type 2 ME49 strain is partially dependent upon MyD88 [7
]. Thus, while there appears to be defects in NFκB activity during early infection, the extent to which this contributes to suppression of cytokines such as IL-12 and TNF-α is unclear.
mediates a rapid but transient activation of MAPK pathways including SAPK/JNK, p38 and ERK1/2 during macrophage infection [100
]. However, subsequent stimulation with LPS fails to result in robust activation normally associated with TLR stimulation [99
] (). Whether Toxoplasma
blocks activation of MAPK through the activity of host or parasite phosphatases, or by other phosphatase-independent mechanisms is not clear. Failure of infected macrophages to respond to LPS restimulation in some ways resembles LPS tolerance. This raises the possibility that T. gondii
induced nonresponsiveness is an endotoxin tolerance phenomenon. Yet, based on several criteria the processes appear to be distinct. Parasite infection induces sustained activation of the MAPK kinase MKK3/6, but, during LPS stimulation of LPS tolerized cells, phosphorylation of MKK3/6 is defective [99
]. In addition, IκBα is resistant to TLR4-induced degradation in LPS tolerized macrophages, whereas in T. gondii
infected cells this molecule undergoes degradation following LPS exposure.
There is evidence that other protozoans target NFκB and MAPK pathways. For example, data suggest that Leishmania
downregulates proinflammatory signaling through induction of SHP-1, a phosphatase that plays a role in deactivation key components of both Jak/Stat and TLR pathways [101
]. Recently, it has been reported that Leishmania
proteases cleave kinases involved in p38 MAPK and NFκB activation [103
]. Infection with T. cruzi
is also reported to induce macrophage TLR nonresponsiveness through induction of host cell phosphatase activity [105
]. Taken together, while it is clear that infection with Toxoplasma
and other protozoans interferes with the ability to respond to TLR ligands, there is not yet a definitive picture of how this is accomplished in any case.