Control of acute toxoplasmosis relies on a potent Th1 cell response that requires IL-12 and IFN-γ production, which are generated through both innate and adaptive responses [21, 22]. It appears that Toxoplasma is unique in that it possesses two mechanisms that trigger IL-12 production in DCs and macrophages [3, 12, 23]. One of these mechanisms is dependent upon the common adaptor protein MyD88, and is likely to involve TLR11 [3, 10, 23]. The other mechanism is dependent upon TgCyp18, which is released by extracellular tachyzoites, triggering IL-12 production through binding to CCR5 . Recently, our group reported that TgCyp18 induced production of NO, TNF-α and IL-12p40 in macrophages, and also up-regulated the production of IFN-γ and IL-6 in these cells . In the present study, the levels of IL-12 in the ascetic fluid of RH-OE-infected CCR5−/− mice were significantly higher than those in RH-GFP-infected CCR5−/− mice, suggesting that cytokine production arose via TgCyp18-dependent and CCR5-independent mechanisms. TgCyp18 stimulated IL-12 production in macrophages  and DCs . Therefore, macrophages and DCs both play a role in IL-12 production in the present study. Further investigations are required to distinguish the relative contributions made by these cells. These results suggest that CCR5-independent accumulation of inflammatory cells at the site of infection might produce higher levels of pro-inflammatory cytokines in CCR5−/− mice.
The ability of T. gondii to attract, invade, and survive inside immune cells (T cells, DCs and macrophages), along with the migratory properties of DCs and macrophages that allow parasite dissemination around the host have been reported previously [7, 24]*. Our results revealed that while T. gondii could infect CD3+, CD11c+, and CD11b+ cells, it exhibited a preference for CD11b+. We observed enhanced recruitment of CD11b+ cells after infection with RH-OE. This chemotactic effect of TgCyp18 was correlated with the ability of RH-OE to increase CCR5 expression levels. Thus, overproduction of TgCyp18 during RH-OE infection enhanced cellular recruitment. Recruitment of CD11b+ cells in CCR5−/− mice infected with RH-OE was also higher than that in RH-GFP-infected mice. Additionally, there was no significant difference in the recruitment of CD11b+ cells between WT and CCR5−/− mice that were infected peritoneally with RH-GFP tachyzoites. Recently, our group demonstrated that recombinant TgCyp18 controlled the in vitro migration of macrophages and lymphocytes in CCR5-dependent and -independent ways . Therefore, the results presented here suggest that the TgCyp18-induced cell migration occurred in a CCR5-independent way in our in vivo experimental model.
Migration of macrophages and lymphocytes to the site of infection would enhance T. gondii invasion into these cells, after which the parasite-infected cells, such as CD11b+ leukocytes, are transported to other organs . Our quantitative PCR analyses revealed that infection with RH-OE resulted in an increased parasitic load in the liver compared with RH-GFP infection. These results suggest that cells recruited by TgCyp18 are used to shuttle the parasite to other organs. In general, chemokines and their receptors play an important role in the migration of immune cells. A previous study showed that an early burst of CCR5 ligand production occurred in the tissue of WT and CCR5−/− mice by day 5 after oral infection with T. gondii strain 76 k cysts . Our present study showed that recombinant TgCyp18 increased the expression levels of CCL5 in macrophages. In addition, significantly higher levels of CCL5 were detected in the peritoneal fluids of CCR5−/− mice infected RH-OE. Moreover, CCL5 levels in the livers of WT mice infected with RH-OE at 5 dpi were higher than those in the other experimental groups. Consistent with our findings, a previous study showed that the parasite numbers in the livers of CCR5−/− mice were higher than those of the C57BL/6 wild-type animals, while the parasite numbers were similar in other organs of the WT and CCR5−/− mice . Therefore, TgCyp18-mediated CCL5 production might contribute to macrophage migration to the site of infection and the transport of T. gondii-infected cells to the liver. Besides CCR5, CCL5 has been shown to interact with other receptors, including CCR3 and CCR1. Therefore, activation of CCR1- and CCR3-signaling may contribute to CCL5-mediated pathology during T. gondii infection. Hence, the chemokines up-regulated in CCR5−/− mice infected with RH-OE may play a crucial role in CCR5-independent macrophage migration.
To test this idea in our study, the expression levels of chemokines related to macrophage migration were investigated. In vitro analysis showed that TgCyp18 increased the expression of CCL6 in a CCR5 independent manner. However, the in vivo data showed that a higher level of CCL6 was observed in the livers of the CCR5−/− mice infected RH-GFP at 3 dpi compared with those infected with RH-OE. Although we do not know the reason for the difference between the in vitro and in vivo data, it is possible that CCL6 expression might have been induced before 3 dpi in the livers of the CCR5−/− mice infected with RH-OE. It is interesting to note that CCL2 expression was slightly increased in macrophages treated with recombinant TgCyp18. Moreover, the expression levels of CCL2 and CXCL10 were significantly higher at 3 dpi in the livers of CCR5−/− mice infected with RH-OE compared with the uninfected mice. Thus, TgCyp18-mediated production of CCL2 and CXCL10 in the liver may trigger transport of T. gondii-infected macrophages via a CCR2 and CXCR3-dependent mechanism, respectively. CCR2−/− mice have profound defects in monocyte recruitment although constitutive trafficking remains unaffected . CCR2−/− mice or CCL2−/− mice failed to recruit Gr1+ inflammatory monocytes, which are required for mucosal resistance to T. gondii, or to control systemic toxoplasmosis by intraperitoneal infection . Furthermore, another group reported that the CXCR3 ligands, CXCL9, CXCL10 and CXCL11, were induced markedly at the levels in the spleen, lung, and liver following infection with T. gondii. Induction of these chemokines was similar in WT and CCR5−/− mice up to day 5 . CXCL10 is required to maintain T-cell populations and to control parasite replication during chronic ocular toxoplasmosis . These results suggest that CCR2 and CCL2, or CXCR3 and its ligands, play a crucial role in cell migration and control of T. gondii infection.
Diana et al.  showed that a T. gondii excreted-secreted antigen induced recruitment and migration of human DCs in a CCR5-dependent fashion. Other studies in mice have reported that T. gondii activates DCs and triggers their migration to the spleen to activate proliferation of T cells, or to potentiate parasite dissemination [25, 33]. TgCyp18 can attract mouse DCs in vitro. CCR5 plays an important role in the migration of intraepithelial CD8+ T cells, and in the regulation of an inflammatory response following T. gondii infection . CCR5 also has a role in the migration of NK cells, with severe deleterious effects observed in infected mice . Thus, it has been shown that increased immune cell migration is involved in the pathogenesis and control of infection with T. gondii. In the present study, based on survival rates, significant differences were not detected in the parasite-challenged (RH-WT, RH-GFP and RH-OE) mice (data not shown). All mice (n = 6) infected intraperitoneally with 1,000 tachyzoites died by 8–9 dpi. All mice (n = 4) infected intraperitoneally with 100 tachyzoites died by 11–15 dpi. Histopathological lesions in livers, spleens and lungs were observed in all mice infected with RH-GFP and RH-OE, but there were no remarkable differences in the severity of the lesions among the experimental groups (Additional file 2: Figure S2). This was probably related to the high virulence of the T. gondii type I strain. In addition, to determine whether macrophages assisted with T. gondii dissemination in the mice, C57BL/6 mice were subject to macrophage depletion by treatment with clodronate liposome, and then challenged with the T. gondii PLK strain (type II). The survival rates of the clodronate-treated and untreated mice were 71% and 43% (n = 7), respectively. Therefore, it appears likely that macrophages assisted with T. gondii dissemination in the mice. However, the pathogenesis of infection with the RH strain is quite different from that of infection with the PLK strain. Hence, further investigations are required to confirm the contribution of TgCyp18 to parasite pathogenesis and the role of macrophages in parasite dissemination.
The recombinant strain (RH-OE) of the parasite expresses TgCyp18 fused to HA. Therefore, it is unclear whether the effects of infection with RH-OE were due to TgCyp18 or HA (or both). To address this, we generated a recombinant T. gondii parasite that expressed the TgCyp18-HA fusion protein as mutants (17GEH19 to 17AAA19 and 149RP150 to 149YV150), which when tested, exhibited reduced interactions with CCR5 (RH-DN, Additional file 3: Figure S3). There was no significant difference in IL-12 production levels in ascites fluid and recruitment of immune cells between the mice infected with RH-GFP and RH-DN (Additional file 4: Figure S4). Therefore, these data suggest that the effects of infection with RH-OE were not due to the HA tag. In addition, the interaction between TgCyp18 and CCR5 played a role in IL-12 production and recruitment of immune cells in the wild type mice.
Taken together, it appears that TgCyp18 might enhance its effects directly through binding with CCR5 and/or another receptor or receptors not yet identified. Regarding TgCyp18-CCR5 dependent or independent responses, there are two explanations that might clarify our observations. First, optimal production of TgCyp18 may under normal circumstances work on CCR5 and/or other receptor(s) to recruit immune cells that produce cytokines. This possibility seems obvious in view of our previous results that showed that TgCyp18 controlled the in vitro migration of macrophages and spleen cells in a CCR5-dependent manner . In contrast, TgCyp18 may initiate cytokine production and macrophage proliferation in a CCR5-independent manner [13, 14]. Second, it is possible that stimulation of host cells with TgCyp18 via CCR5 and/or other receptor(s) could trigger expression of chemokine receptors and its ligands for cell migration. Increased CCL5 levels in the livers of the wild-type mice infected with RH-OE parasites indicates that parasite migration to this organ occurred in a TgCyp18- and CCR5-dependent manner. Furthermore, parasite migration, which occurred in a CCR5-independent and TgCyp18-dependent way, can be explained by the higher levels of CCL2 and CXCL10 in the liver and CCL5 in the ascites fluid of CCR5−/− mice infected with RH-OE. Thus, the present results suggest that TgCyp18 has the ability to enhance host-cell migration via CCL5 and parasite dissemination by CCL2 and CXCL10 in a CCR5-independent manner.