We investigated the role of IFN-γ in expression of chemokines at both mRNA and protein levels in the brain during chronic infection with type II T. gondii in BALB/c and IFN-γ−/− mice. Using semiquantitative RT-PCR, we observed significant increases of mRNA for CXCL9/MIG, CXCL10/IP-10, CXCL11/I-TAC, CCL2/MCP-1, CCL3/MIP-1α, and CCL5/RANTES in the brains of infected wild-type mice, as compared to uninfected wild-type animals. Among these chemokines, CXCL9/MIG, CXCL10/IP-10, and CCL5/RANTES were relatively most abundant. Quantification of the chemokine proteins demonstrated that CXCL9/MIG, CXCL10/IP-10, and CCL5/RANTES are those predominantly expressed in the brains of chronically infected wild-type BALB/c mice. To our knowledge, this is the first evidence of expression of chemokine proteins in the brain of T. gondii infected hosts.
In contrast to wild-type BALB/c mice, we detected only low levels of all the chemokine proteins in the brains of infected IFN-γ−/− mice. Levels of CXCL9/MIG, CXCL10/IP-10, and CCL5/RANTES proteins, which are most abundant in the brain of infected wild-type animals, were limited in the IFN-γ−/− mice only at 0.91%, 3.65%, and 9.1% of the levels in the wild-type animals, respectively. These results correlated with mRNA levels of each of these chemokines (<0.1%, 8.3%, and 27.1%, respectively) measured by real-time RT-PCR. Thus, IFN-γ is crucial for inducing expression of these 3 chemokines in both mRNA and proteins in the brain during the chronic stage of infection with T. gondii. Because of the time course (the chronic stage of infection) of this study, in addition to IFN-γ itself, mediators induced by IFN-γ could also be involved in expression of these chemokines. However, regardless of a direct or indirect effect, it is clear that IFN-γ plays a critical role in expression of CXCL9/MIG, CXCL10/IP-10, and CCL5/RANTES in the brains of mice chronically infected with T. gondii. The importance of IFN-γ in chemokine expression in the brain during chronic infection with this parasite was not reported before.
It was previously reported that TNF-α can induce expression of CXCL10/IP-10 and CCL5/RANTES in astrocytes in vitro
(Barnes and others 1996
; Salmaggi and others 2002
). We detected TNF-α mRNA in the brains of infected IFN-γ−/−
mice as high as a half of the levels expressed in infected wild-type BALB/c mice. Therefore, this TNF-α expression is most likely a reason for the induction of expression of CXCL10/IP-10 and CCL5/RANTES in the brains of infected IFN-γ−/−
mice, although their expression levels are less than those in infected wild-type animals. In support of this possibility, the amount of TNF-α mRNA in each individual of infected IFN-γ−/−
mice correlated well with the amounts of CXCL10/IP-10 and CCL5/RANTES mRNA expressed in each individual (data not shown).
In relation to our finding on the importance of IFN-γ in expression of chemokine mRNA and proteins in the brains during chronic infection with T. gondii
, Strack and others (2002
) previously reported that IFN-γ−/−
mice failed to express mRNA for CXCL9/MIG, CXCL10/IP-10, and CCL5/RANTES and expressed reduced levels of mRNA for CCL2/MCP-1, CCL3/MIP-1α, and CCL4/MIP-β in their brains during acute systemic toxoplasmosis (10 days after infection). Thus, along with the finding in this study, IFN-γ plays an important role in inducing chemokine expression during both the acute and chronic stages of T. gondii
infection, whereas its effects on the expression seems to differ between the acute and chronic stages of infection.
In an acute systemic toxoplasmosis model, CXCL10/IP-10 expression was observed in astrocytes, whereas CXCL9/MIG and CCL5/RANTES expression was in microglia (Strack and others 2002
). These glial cells could be the source of these chemokines during the chronic stage of infection as well. In vitro
studies demonstrated that human and simian astrocytes express CXCL9/MIG and CCL5, in addition to CXCL10/IP-10, following stimulation with IFN-γ plus TNF-α (Croitoru-Lamoury and others 2003
). Since both IFN-γ and TNF-α are expressed in the brains of mice chronically infected with T. gondii
, it is possible that astrocytes, in addition to microglia, produce these chemokines in their brain. Active proliferation of tachyzoites does not occur in the brains of genetically resistant BALB/c mice during the chronic stage of infection. In addition, these animals have only small numbers (less than a few hundred in the entire brain) of tissue cysts, the dormant stage of the parasite, in their brains (Suzuki and others 1993
; Brown and others 1995
). Therefore, a majority of the cells producing the chemokines in the brains of chronically infected BALB/c mice would probably not be those infected with the parasite.
Our previous study demonstrated an importance of IFN-γ for recruiting CD8+
immune T cells into the brains of chronically infected BALB/c mice (Wang and others 2007
). CXCR3, the receptor for CXCL9/MIG and CXCL10/IP-10, is expressed predominantly on activated T cells, and this chemokine receptor is considered to play the primary role in recruitment of effector T cells into the site of a type I immune response. Since IFN-γ production by T cells, especially CD8+
T cells, that migrated into the brain is required for maintaining the latency of chronic T. gondii
infection in the brain and genetic resistance of BALB/c mice to development of TE (Wang and others 2004
), IFN-γ-mediated expression of CXCL9/MIG and CXCL10/IP-10 in the brains of infected BALB/c mice, as revealed in the present study, could play a crucial role in recruitment of IFN-γ-producing effector T cells into the brain for prevention of TE during the chronic stage of infection.
CCL5/RANTES, another chemokine abundantly expressed in the brain of chronically infected BALB/c mice, is 1 of 3 ligands that bind to CCR5. In addition to CXCR3, CCR5 is preferentially expressed on TH
1 cells and plays an important role in infiltration of these T cells in TH
1-type reactions (Moser and Loetscher 2001
). CCR5 is also expressed on macrophages, which are important effector cells activated by IFN-γ to prevent proliferation of T. gondii
tachyzoites (Suzuki and others 1988
). Therefore, expression of CCL5/RANTES in combination with CXCL9/MIG and CXCL10/IP-10 could play an important role in recruiting T cells to maintain a type I immune response and facilitating migration of effector macrophages into the brain to control the parasite.
CXCL9/MIG, CXCL10/IP-10, and CCL5/RANTES play nonredundant roles in resistance against viral infection in the brain (Liu and others 2000
; Ure and others 2005
). During the acute stage of T. gondii
infection, CXCL10/IP-10 plays an important role in inducing massive influx of T cells into the lungs and livers and controlling the parasite in these organs (Khan and others 2000
). CCR5 is important for natural killer (NK) cell trafficking into the spleen and liver and host survival after acute acquired infection (Khan and others 2006
), whereas NK cells do not appear to be required for prevention of TE during the later stage of infection (Kang and Suzuki 2001
). Therefore, CXCL9/MIG, CXCL10/IP-10, and CCL5/RANTES could induce migration of immune cells into different organs depending on the stages of infection, and the cell populations that migrate into the organs and control the parasite could be different between the stages. During the chronic stage of infection with T. gondii
, CXCL9/MIG, CXCL10/IP-10, and CCL5/RANTES may play important roles in recruiting immune T cells and macrophages into the brain to maintain the latency of infection and to prevent TE.