UNC93B1 is a critical mediator of the translocation of nucleotide-sensing TLR3, TLR7 and TLR9 from the ER to endolysosomes 
. Here, we tested the 3d mouse, which has a non-functional UNC93B1 
, to evaluate the combined role of nucleotide sensing TLRs in controlling initial activation of innate immunity and host resistance to infection with T. gondii
. Despite the fact that none of single TLR3, TLR7 or TLR9 knockout yield an altered phenotype on cytokine response or enhanced susceptibility, we found that 3d mice are extremely susceptible to infection with T. gondii.
Therefore, our results raise the possibility that combined action of nucleotide sensing TLRs is critical for host resistance to T. gondii
Much to our surprise, the MyD88/TRIF null mice, which are devoid of all TLR functions and show impaired production of pro-inflammatory cytokines when infected with T. gondii
, were consistently more resistant to infection than the 3d mice. Furthermore, except for IL-12, 3d mice infected with T. gondii
mounted a normal systemic pro-inflammatory response, while the MyD88/TRIF double knockouts did not, indicating that the immunological response to infection was fundamentally different. Nevertheless, animals bearing UNC93B1 mutation succumbed to infection as a result of unchecked tachyzoite replication, similar to IFNγ−/−
. Thus, while we cannot exclude that the combined action of intracellular TLR 3, 7 and 9 contributes to host resistance against T. gondii
, our hypothesis is that UNC93B1 also mediates host resistance against T. gondii
through an additional mechanism, which is TLR-independent.
Considering the UNC93B1 involvement on antigen presentation 
, we first investigated whether antigen presenting cells (APCs), CD4+
T, and CD8+
T lymphocytes were properly activated in 3d mice. Our results show that production of IL-12 as well as expression of activation markers by APCs was significantly impaired in 3d mice infected with T. gondii.
Intriguingly, the IFNγ levels were similar in the sera, splenocyte cultures and peritoneal cavity, when comparing 3d and WT mice at day 8 post-infection with T. gondii
. While the percentage of T cells producing IFNγ in splenocytes from infected 3d was comparable to infected C57BL/6 mice, the total numbers of IFNγ producing-CD4+ T as well as -CD8+ T cells were lower in de 3d mice. Regardless, mice deficient in CD8+
T cells or in the so-called transporter associated with antigen processing (TAP-1) protein, while more susceptible to infection with T. gondii
, often survive 30–40 days post-infection 
. Thus, our data suggest that defective antigen cross-presentation and CD8+
T cell activation are not the primary events accounting for the extreme susceptibility of 3d mice to T. gondii
Importantly, our in vivo
experiments suggest that UNC93B1 is an important mediator of IL-12 production during T. gondii
infection. We also addressed this question in vitro
and observed that exposure of macrophages from 3d or MyD88−/−
mice to live ME49 tachyzoites resulted in impaired production of IL-12, as compared to macrophages from WT mice. Since IL-12 is a key mediator of IFNγ production during T. gondii
, it is surprising that IFNγ responses, as discussed above, were close to normal in the infected 3d mice. Therefore, we performed experiments at earlier time points and observed that production of IL-12 and IFNγ was significantly impaired in the peritoneal cavity and peritoneal cavity/spleens from 3d mice at 3 and 5 days post-infection, respectively. These findings could be explained as a result of combined deficiency of nucleotide sensing-TLRs, since no phenotype is observed in each of the single TLR3, TLR7 or TLR9 knockout mice.
Consistently, experiments performed in our laboratory and elsewhere 
demonstrate that despite of severe impairment IL-12 production in MyD88−/−
mice, IFNγ is still produced at 8 days post-infection, and yet, mice are highly susceptible to ME-49 infection. Similarly, different studies demonstrate that except for IL-12, infection with the highly virulent RH strain of T. gondii
elicits elevated levels of pro-inflammatory cytokines, including IFNγ 
. However due to the inherent ability of RH parasites to rapidly replicate and disseminate, infected animals are still unable to control parasite burden and die during the acute phase of infection 
Even though the observed defect on IL-12/IFNγ axis seems to be sufficient to render animals more susceptible to T. gondii
, our results clearly indicate that an additional TLR independent-function mediated by UNC93B1 contributes for the extreme susceptibility of 3d mice, when compared to MyD88−/−
or MyD88/TRIF double knockouts infected with T. gondii
. Notably, from in vitro
and in vivo
experiments we had no evidence that cells from 3d mice have a defect in responding to IFNγ. Thus, we next investigated the ability of host cells from UNC93B1 mutant mice to control parasite replication. The fate of T. gondii
inside the host cell relies upon the mechanism of entry. Passive internalization by phagocytosis directs parasites to the lysosomal compartment, leading to tachyzoite elimination 
. Upon active invasion, T. gondii
establishes itself in the parasitophorous vacuole 
, which avoids fusion with lysosomes, allowing parasite survival. Interestingly, we found that UNC93B1 is recruited from the ER to the parasitophorous vacuole (PV), rather than to the expected endolysosomal compartment 
In spite of being considered a non-fusogenic compartment, few host cell proteins are found at the membrane of parasitophorous vacuole containing tachyzoites. Specific proteins appear to be selectively recruited from the host cell plasma membrane 
, or after host cell activation 
. In addition, the ER is known to be in close contact with the parasitophorous vacuole membrane 
, and fusion between the ER and parasitophorous vacuole containing live parasites has been demonstrated 
. Notably, UNC93B1 was shown to translocate from ER to the endolysosomal compartment upon cell stimulation with TLR agonists 
. Notwithstanding, here we show that UNC93B1 is recruited from the ER to the parasitophorous vacuoles. It is likely that the recruitment of UNC93B1 to the parasitophorous vacuole membrane occurs during the process of ER fusion, given that UNC93B1 is an ER resident protein 
. The transfer of ER proteins to the parasitophorous vacuole membrane seems to be selective, since neither the mutant form of UNC93B1 nor TLR9 (not shown) were found to be enriched around the parasitophorous vacuole. Dissociation of the intracellular traffic of UNC93B1 and TLR9 has also been suggested by Ewald and co-workers 
, as they observed that forced expression of UNC93B1 at the plasma membrane was not accompanied by relocation of TLR9. Certainly, the process that involves selection or exclusion of specific host proteins, including UNC93B1 and nucleotide sensing TLRs, to the parasitophorous vacuole membrane, is likely to be a key event in the successful establishment of parasitism, and remains to be elucidated.
Most of the mechanisms involved in the control of intracellular replication of T. gondii
have been studied in IFNγ-activated macrophages. For example the downstream effects of GTPases 
, production of reactive nitrogen intermediates 
, tryptophan degradation in human cells 
or autophagy 
are all IFNγ-inducible mechanisms involved in controlling and/or killing of T. gondii
replication. Markedly, we found that in vitro
, macrophages from 3d mice present a normal response and are perfectly able to control tachyzoite replication when activated with IFNγ. Consistently, the production of reactive nitrogen intermediates as well as and expression and translocation of IFNγ-inducible GTPases (i.e.
Irga6, Irgb6, Irgm1 and Irgm3) or formation of autophagic vacuoles (data not shown) are not impaired in macrophages from 3d mice activated with IFNγ.
Remarkably, our results demonstrate an uncontrolled parasite replication in macrophages from 3d mice infected in vivo
with T. gondii
, despite an unimpaired IFNγ response. Further, we found that in vitro
the 3d mutation renders non-activated macrophages more susceptible to intracellular tachyzoite replication. Even though the difference in parasite numbers is modest, when comparing macrophages from WT and 3d mice, it may reflect large differences in vivo
, where multiple rounds of parasite replication during a long period of time will result in exponential parasite growth. To support our interpretation, other studies also show that small but significant differences in parasite replication in vitro,
reflects dramatic differences in parasite growth and virulence in vivo 
. It is difficult to imagine how this phenomenon could be related to the effects of the UNC93B1 mutation on TLR signaling, since the rates of parasite replication in non-activated macrophages from MyD88/TRIF null mice were similar to that observed in the same cells derived from wild-type mice. Thus, UNC93B1 effects on parasite control appear to be independent of what are thought important immune mediators of host resistance to T. gondii
, such as TLRs, IFNγ and TNFα.
To establish itself inside a host cell T. gondii
has to acquire metabolites from intracellular stores. Indeed, UNC93B1 is a distant ortholog to an ion transporter from Caenorhabditis elegans 
. Despite this homology, a similar function has not been described for UNC93B1 in mammals 
. Alternatively, nutrient can be acquired from channels present at the parasitophorous vacuole membrane, which allow free diffusion of small metabolites up to 1300 Da 
; lipids may be acquired through the closely apposed mitochondrial and ER membranes 
; and parasite seems to exploit the host endolysosomal system via sequestration of host organelles into invaginations present at the parasitophorous vacuole membrane 
. Therefore, it is also possible that UNC93B1 regulates metabolite/nutrient acquisition by T. gondii
tachyzoites, and hence interferes with parasite replication.
In conclusion, our study reveals a critical anti-parasitic role for UNC93B1. This role appears to involved in at least two steps: (i) control of IL-12 and early IFNγ response, which may be a result of combined TLR3/TLR7/TLR9 deficiency; and (ii) UNC93B1 enrichment in the membranes surrounding the parasitophorous vacuole containing T. gondii tachyzoites, which mediates control of parasite growth in a TLR- and IFNγ-independent manner. Altogether our results indicate that UNC93B1 plays a critical role on innate immune response and host resistance to T. gondii infection.