Our data presented here identifies a novel role for the T. gondii enzyme Prx in modulating macrophage effector functions by promoting alternative activation of macrophages and enhanced parasite replication. Exposure to rTgPrx promoted elevated arginase-1 enzyme activity and mRNA levels. In addition the markers of alternative activation Ym1 and FIZZ were also upregulated at the mRNA level. The increased expression of these genes is indicative of alternative activation . Additionally IL-10 synthesis was also increased in cells treated with rTgPrx, this is a common phenotype of cells exposed to helminth molecules undergoing alternative activation . We determined that the increased arginase activity caused by rTgPrx was dependent on STAT6 signalling, a key transcription factor in the initiation of Th2 responses downstream of IL-4/IL-13 signalling. However this was the only effect of rTgPrx that was STAT6-dependent, revealing differential control mechanisms of genes involved in alternative activation by rTgPrx. This is in contrast with traditional alternative activated macrophages generated by IL-4 conditioning, whereby expression of arg-1, Ym1, and FIZZ is conditional on STAT6 activation. More significantly we found that rTgPrx alters host cell ROS status suggesting that the effects of rTgPrx is reliant on intrinsic enzyme activity interfering in the host cell redox pathway to alter intracellular immunity signalling. We confirmed the need for intrinsic enzyme activity by proteinase K treatment of rTgPrx prior to experiments resulting in a loss of anti-oxidant function (additional file 3). This alteration may be responsible for our findings of reduced caspase-1 activation and IL-1β levels. Recently a report has suggested such a link between alterations in cellular redox status and activation of the NLRP3/caspase-1/IL-1β inflammasome . Our final finding of increased parasite replication following pre-treatment of cells with rTgPrx indicates its role as an immunomodulator by negatively acting on anti-parasitic macrophage functions.
The function of alternatively activated macrophages within helminth parasitic infections is widely studied and they have been reported to have a number of roles dependent on model studied, including providing protection against Heligosomodies polygyrus infection , preventing immunopathology during S. mansoni infection , and controlling Th2 immunity [27, 28]. However in terms of protozoan infections the role of AAM is an unresolved issue, due to the cytkine milieu that occurs during this class of infection, i.e. predominately "Th1" cytokines IL-12/IFN-γ, one might expect classically activated macrophages to dominate. There are however reports of AAM during infection with Trypansome spp., infection of resistant C57 mice with T. conglesses resulting in a changing CAM to AAM population as infection progressed . This change is thought to reflect a changing need to control parasitemia initially and later to dampen inflammation and protect the host. Ultimately this switch favours host and parasite survival. To our knowledge the only work addressing the issue of AAM in T. gondii infection related to mice with a dominant negative mutation in the IFN-γ receptor in macrophage-lineage cells, thus making these cells incapable of activating anti-protozoan effector functions . These mice despite showing the expected immune response to T. gondii, were unable to control parasitemia specifically within macrophages and as a consequence displayed reduced survival. Using macrophages unable to alternatively activate, non-responsive to IL-4, in the non-healing model of L. major infection in Balb/c mice, there was a Th2 response generated but effective control of parasite numbers within macrophages, again reflecting ineffectiveness of AAM in terms of controlling protozoan replication . Taken together these results would suggest that while the presence of AAM could lead to enhanced mortality during acute infection with some protozoans, a switch to an AAM phenotype might lead to control of immunopathology and benefit both host and parasite long-term. This theory may not fit with the known course of disease following infection of ovine hosts with T. gondii. During pregnancy a reactivation of tissue cysts results in massive parasite replication and pathology at the foetal-maternal interface resulting in abortion . The prevalent immune environment during pregnancy would favour AAM or indeed regulatory macrophages, favouring production of IL-10 and less pro-inflammatory cytokines, similar to the kind seen in our experiments. However our results certainly raise the possibility that rTgPrx may represent a previously unidentified immunomodulator.
The most pertinent questions surrounding the function of rTgPrx regards the trafficking of rTgPrx both inside the parasite and host. Analysis of the protein sequence reveals no secretion signal peptides and one must assume the enzyme is not exported from the parasite. However when initially cloned rTgPrx was found to be present in both the extra- and intra-cellular forms of the parasite. Its distribution within the tachyzoite is limited and was not found to be present in the parasitophorous vacuole or parasitophorous vacuole membrane . Indeed immunolocalisation shows that rTgPrx is present in the parasite nucleus . It is thought that under oxidative stress parasite signalling pathways lead to Prx translocating to the nucleus and forming a complex the KMTox, a histone lysine methyltransferase, to regulate gene expression. The question as to how rTgPrx acts on host cells will require further experimentation. However should a parasite become damaged upon entry to the host cell or as a result of oxidative damage, it is not inconceivable that cytosolic rTgPrx make "leak" into the host cell environment.
To conclude our results clearly show that an enzyme, rTgPrx, derived from T. gondii promotes AAM and alongside IL-10 secretion via STAT6 dependent and independent mechanisms, whilst simultaneously downregulating IL-1β production via caspase-1. Negative modulation of the inflammasome has recently been reviewed in the context of bacterial and viral infection , however results presented here would suggest exploration of this phenomena in the context of T. gondii infection warrants investigation. The functional relevance of this effect has only been explored in vitro in the context of parasite replication which is enhanced after pre-treatment of cells with rTgPrx. However the role of this protein and rTgPrx induced AAM during T. gondii infection warrants further in vivo investigation, the most suitable method being production of a parasite bearing a mutant Prx.