LRG-47 and IRG-47 belong to a family of IFN-γ–induced proteins whose expression is increased to very high levels in mice after infection with numerous pathogens, including T. gondii, L. monocytogenes, and MCMV (). To determine the roles that LRG-47 and IRG-47 might play in mediating IFN-γ–stimulated resistance to these pathogens, we used gene targeting to create mice that lacked expression of the two proteins (), and then assessed their ability to restrict the three infections. In absence of infection, the LRG-47 and IRG-47–deficient mice displayed no obvious abnormalities; they were produced in normal numbers, and necropsies revealed no major alterations in tissue architecture (data not shown). In addition, FACS® analysis of splenocytes from adult mice revealed no changes in the development of T cell, B cell, macrophage, and NK cells (data not shown). However, when challenged with the three model pathogens, the mice displayed profound, but selective, losses in host resistance.
Figure 1 Induction of LRG-47 and IRG-47 expression in response to different pathogens. Pairs of mice were inoculated as indicated with 20 cysts T. gondii for 8 d, 1,000 CFU L. monocytogenes for 5 d, or 5 × 104 PFU MCMV for 36 h, or were left uninfected (more ...)
Figure 2 Gene targeting to create LRG-47– and IRG-47–deficient mice. As described in detail in the Materials and Methods, standard gene targeting techniques were used to generate mice that lack production of LRG-47 and IRG-47. Western blotting (more ...)
We began by examining the susceptibility of the mice to the intracellular protozoan parasite T. gondii.
Infection with T. gondii
is characterized by an acute phase in which the rapidly proliferating form, or tachyzoite, disseminates throughout the host, followed by a chronic phase in which a dormant form, or bradyzoite, inhabits mainly central nervous tissue and muscle 21
. IFN-γ is absolutely required for control of both phases 1321
. After invasion of the host cell, T. gondii
resides in a parasitophorous vacuole that resists interaction with the endocytic machinery of the cell, and consequently provides the organism with a safe environment in which to replicate 22
. LRG-47–deficient, IRG-47–deficient, and wild-type mice were inoculated intraperitoneally with 20 cysts of T. gondii
, and their responses were assessed. Similar to what has been shown previously for IGTP-deficient mice 12
, LRG-47–deficient mice displayed a complete loss of resistance to the parasite during the acute phase of infection, dying between days 9 and 11 after inoculation ( A), the same time frame in which IFN-γ–deficient mice succumb to the infection 13
. When the mice were examined 5 d after infection, there was a marked increase in the number of T. gondii
infected cells in the peritoneum of the LRG-47–deficient mice ( B), comparable to that seen in IFN-γ–deficient mice 13
, which indicated that the mice were not able to suppress replication of the parasite. Also at 5 d after infection, IFN-γ and IL-12 production was robust ( C), indicating that loss of resistance was not due to lack of production of the two cytokines. The modestly elevated levels of the two cytokines in the LRG-47–deficient mice were probably due to the unattenuated replication of the parasite. Importantly, decreased resistance to T. gondii
in the LRG-47–deficient mice was not a result of decreased IGTP production, given that IGTP levels in splenocytes increased to high levels after T. gondii
infection in vivo (data not shown), and IGTP expression was increased normally in LRG-47–deficient fibroblast cultures after stimulation with IFN-γ ( A). Therefore, IGTP and LRG-47 are independent factors that are both critically important for normal clearance of acute T. gondii
Figure 3 Acute loss of resistance to T. gondii in LRG-47–deficient mice. The indicated strains were inoculated intraperitoneally with 20 cysts T. gondii, and their ability to restrict the infection was assessed. (A) Wild-type (n = 6) and LRG-47–deficient (more ...)
In contrast to the IGTP-deficient and LRG-47–deficient mice, IRG-47–deficient mice displayed only marginally reduced resistance to T. gondii (). After challenge with 20 cysts of the parasite, 67% of the IRG-47–deficient mice died, with death occurring mainly during the chronic phase between days 10 and 47 after infection (). The burden of T. gondii cysts in the brains of IRG-47–deficient mice correlated with the partial decrease in survival in the chronic phase of infection: at 33 d after infection there were 2,575 ± 1,237 cysts per brain in IRG-47–deficient mice vs. 3,300 ± 1,387 in wild-type mice, while at 57 d there were 1,447 ± 654 in IRG-47–deficient mice vs. 913 ± 228 in wild-type mice. IFN-γ production was normal in IRG-deficient mice, with peritoneal exudate cells from IRG-47–deficient mice producing 5,164 ± 1,153 pg/ml IFN-γ after exposure to soluble T. gondii tachyzoite extract, STAg, compared with 4,558 ± 3,011 pg/ ml from wild-type cells. Thus, as opposed to LRG-47 and IGTP, IRG-47 plays only a modest role in restricting T. gondii infections, and this does not become apparent until the chronic phase of infection. The differing responses of the mice to T. gondii infections reflect the subdivision of the IGTP protein family into two subfamilies based on primary sequence homology, with one subfamily containing LRG-47 and IGTP, and the second IRG-47.
Marginal loss of resistance to T. gondii in IRG-47–deficient mice. Wild-type (n = 17) and IRG-47–deficient mice (n = 15) were monitored for their survival for 60 d. Shown are the cumulative results of two experiments. KO, knockout.
Next, the mice were challenged with L. monocytogenes
, a gram-positive bacterium that produces an acute infection. L. monocytogenes
is an intracellular bacterium that resides in a vacuole briefly after penetration of the host cell, but thereafter lyses the vacuole to release itself into the cytosol where it replicates 23
. IFN-γ signaling is critical for normal restriction of L. monocytogenes 15
, but IGTP is not required for normal resistance 12
. To determine whether LRG-47 and IRG-47 are required, mice that lack their expression were infected with 1,000 CFUs of L. monocytogenes
and then monitored for their responses ( and ). Surprisingly, LRG-47–deficient mice succumbed rapidly, displaying uniform death by 5 d, paralleling that seen in IFN-γ–deficient mice 1215
( A). Decreased survival in the LRG-47–deficient mice correlated with greatly increased bacterial burdens in the liver and spleen at 3 d after infection ( B). Also in the LRG-47–deficient mice, IFN-γ production was equivalent to that of wild-type mice, while IL-12 production was elevated ( C).
Figure 5 Marked loss of resistance to L. monocytogenes in LRG-47–deficient mice. The indicated strains were inoculated intraperitoneally with 1,000 CFU L. monocytogenes, and their ability to restrict the infection was assessed. (A) Wild-type (n = 4) and (more ...)
Figure 6 Normal resistance to L. monocytogenes in IRG-47–deficient mice. (A) Wild-type (WT; n = 6), IFN-γ–deficient (n = 6), and IRG-47–deficient mice (n = 6) were monitored for their survival for 40 d. KO, knockout. (B) (more ...)
In contrast to the LRG-47–deficient mice, the IRG-47–deficient mice showed no adverse effects and 100% survival after L. monocytogenes
infection ( A). In addition, the bacterial loads in the liver and spleen of IRG-47–deficient mice were equivalent to those in wild-type mice ( B). Thus, LRG-47 plays a central role in mediating IFN-γ–induced clearance of L. monocytogenes
, whereas IRG-47 and IGTP 12
Finally, the response of the mice to MCMV was characterized. MCMV is a double-stranded DNA herpes virus that, in an immunocompetent host, establishes a latent infection; however, absence of IFN-γ signaling leads to an acute infection with increased viral loads and mortality 24
. LRG-47–deficient and IRG-47–deficient mice were inoculated with MCMV, and at 3 d after inoculation, the loads of MCMV in tissues of the mice were determined. In both spleen and liver, comparable numbers of viral plaque-forming units were detected in wild-type, LRG-47–deficient, and IRG-47–deficient mice (data not shown). Similar results have been reported previously for IGTP-deficient mice 12
. Therefore, LRG-47, IRG-47, and IGTP are not critical factors for defense against MCMV.
Taken together, our data demonstrate that LRG-47 and IRG-47 are members of a protein family that plays a central role in the IFN-γ–mediated clearance of infection, with each member of the family supporting host resistance to a different spectrum of pathogens (). Because each of the genes is expressed at high levels after infection, their differential roles in host resistance are likely to relate to distinct molecular functions. Although LRG-47, IRG-47, and the related proteins are expressed in hematopoietic and nonhematopoietic cells, there is no evidence to suggest that they modulate classical immune functions, given that LRG-47–, IRG-47–, and IGTP-deficient macrophages produce normal levels of nitric oxide and TNF-α (data not shown), and IGTP-deficient CD8 T cells and NK cells exhibit normal cytotoxic functions 12
. Rather, it seems likely that the proteins act within the host cell to undermine survival of invading pathogens. Considering their purported roles as regulators of protein expression or trafficking 5
, it is possible that LRG-47 and IRG-47 may function by altering trafficking to host cell vacuoles that contain T. gondii
or L. monocytogenes
, thereby affecting vacuole acidification and maturation, and compromising survival of the pathogen. Parallels can be drawn with some members of the rab family of GTP-binding proteins that are present in the endosomal compartment, as in vitro data suggest that they may regulate vesicular trafficking to intracellular pathogens 2526.
The studies presented here suggest that another family of GTP-binding proteins in a compartment more distal to the vacuole, the endoplasmic reticulum (ER), plays a critical role in governing the fate of microbes in IFN-γ–activated cells.
Differential Loss of Host Resistance to Representative Pathogens among Mice Lacking IGTP Family Proteins