Inactivation of LRG-47 and IRG-47 reveals a family of interferon gamma-inducible genes with essential, pathogen-specific roles in resistance to infection - PubMed (original) (raw)

Inactivation of LRG-47 and IRG-47 reveals a family of interferon gamma-inducible genes with essential, pathogen-specific roles in resistance to infection

C M Collazo et al. J Exp Med. 2001.

Abstract

The cytokine interferon (IFN)-gamma regulates immune clearance of parasitic, bacterial, and viral infections; however, the underlying mechanisms are poorly understood. Recently, a family of IFN-gamma-induced genes has been identified that encode 48-kD GTP-binding proteins that localize to the endoplasmic reticulum of cells. The prototype of this family, IGTP, has been shown to be required for host defense against acute infections with the protozoan parasite Toxoplasma gondii, but not for normal clearance of the bacterium Listeria monocytogenes and murine cytomegalovirus (MCMV). To determine whether other members of the gene family also play important roles in immune defense, we generated mice that lacked expression of the genes LRG-47 and IRG-47, and examined their responses to representative pathogens. After infection with T. gondii, LRG-47-deficient mice succumbed uniformly and rapidly during the acute phase of the infection; in contrast, IRG-47-deficient mice displayed only partially decreased resistance that was not manifested until the chronic phase. After infection with L. monocytogenes, LRG-47-deficient mice exhibited a profound loss of resistance, whereas IRG-47-deficient mice exhibited completely normal resistance. In addition, both strains displayed normal clearance of MCMV. Thus, LRG-47 and IRG-47 have vital, but distinct roles in immune defense against protozoan and bacterial infections.

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Figures

Figure 1

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 (control). Total RNA was prepared from liver and used for sequential Northern blotting with LRG-47, IRG-47, IGTP, and GAPDH probes. Positions of the major ribosomal RNA species are indicated.

Figure 3

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 mice (n = 6) were monitored for their survival for 40 d. KO, knockout. (B) Peritoneal exudate cells from wild-type (WT; n = 3), IFN-γ–deficient (n = 3), and LRG-47–deficient mice (n = 4) were isolated at 5 d after infection, and the presence of intracellular T. gondii was determined microscopically. (C) Sera were isolated from wild-type (n = 2) and LRG-47–deficient mice (n = 4) at 5 d after infection and used for IFN-γ and IL-12 determination by ELISA. A–C are representative results of two experiments.

Figure 3

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 mice (n = 6) were monitored for their survival for 40 d. KO, knockout. (B) Peritoneal exudate cells from wild-type (WT; n = 3), IFN-γ–deficient (n = 3), and LRG-47–deficient mice (n = 4) were isolated at 5 d after infection, and the presence of intracellular T. gondii was determined microscopically. (C) Sera were isolated from wild-type (n = 2) and LRG-47–deficient mice (n = 4) at 5 d after infection and used for IFN-γ and IL-12 determination by ELISA. A–C are representative results of two experiments.

Figure 3

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 mice (n = 6) were monitored for their survival for 40 d. KO, knockout. (B) Peritoneal exudate cells from wild-type (WT; n = 3), IFN-γ–deficient (n = 3), and LRG-47–deficient mice (n = 4) were isolated at 5 d after infection, and the presence of intracellular T. gondii was determined microscopically. (C) Sera were isolated from wild-type (n = 2) and LRG-47–deficient mice (n = 4) at 5 d after infection and used for IFN-γ and IL-12 determination by ELISA. A–C are representative results of two experiments.

Figure 2

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 was then used to verify absence of protein expression. (A) Embryonic fibroblasts from wild-type (+/+) or LRG-47–deficient (−/−) mice were exposed to control conditions or 100 U/ml IFN-γ for 15 h. Lysates were prepared from the cells, resolved by 10% SDS-PAGE, and used for sequential Western blotting with anti–LRG-47 and anti-IGTP antisera. (B) Spleen and thymus were isolated from wild-type (+/+) or IRG-47–deficient (−/−) mice. Lysates were prepared, resolved by 10% SDS-PAGE, and used for Western blotting with anti–IRG-47 antisera. Expression in IRG-47–deficient fibroblasts could not be assessed because of cross-reacting bands in the fibroblast lysates that were recognized by the anti–IRG-47 antisera.

Figure 2

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 was then used to verify absence of protein expression. (A) Embryonic fibroblasts from wild-type (+/+) or LRG-47–deficient (−/−) mice were exposed to control conditions or 100 U/ml IFN-γ for 15 h. Lysates were prepared from the cells, resolved by 10% SDS-PAGE, and used for sequential Western blotting with anti–LRG-47 and anti-IGTP antisera. (B) Spleen and thymus were isolated from wild-type (+/+) or IRG-47–deficient (−/−) mice. Lysates were prepared, resolved by 10% SDS-PAGE, and used for Western blotting with anti–IRG-47 antisera. Expression in IRG-47–deficient fibroblasts could not be assessed because of cross-reacting bands in the fibroblast lysates that were recognized by the anti–IRG-47 antisera.

Figure 5

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 LRG-47–deficient mice (n = 6) were monitored for their survival for 40 d. KO, knockout. (B) Spleen and liver of wild-type (WT; n = 7), IFN-γ–deficient (n = 7), and LRG-47–deficient mice (n = 9) were isolated at 3 d after infection, and the numbers of bacteria present was determined. Statistical analysis for wild-type vs. LRG-47–deficient mice: spleen, P = 0.003; liver, P = 0.05. (C) Sera were isolated from wild-type (n = 3) and LRG-47–deficient mice (n = 5) at 3 d after infection and used for IFN-γ and IL-12 determination by ELISA. A–C are representative results from two experiments.

Figure 5

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 LRG-47–deficient mice (n = 6) were monitored for their survival for 40 d. KO, knockout. (B) Spleen and liver of wild-type (WT; n = 7), IFN-γ–deficient (n = 7), and LRG-47–deficient mice (n = 9) were isolated at 3 d after infection, and the numbers of bacteria present was determined. Statistical analysis for wild-type vs. LRG-47–deficient mice: spleen, P = 0.003; liver, P = 0.05. (C) Sera were isolated from wild-type (n = 3) and LRG-47–deficient mice (n = 5) at 3 d after infection and used for IFN-γ and IL-12 determination by ELISA. A–C are representative results from two experiments.

Figure 5

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 LRG-47–deficient mice (n = 6) were monitored for their survival for 40 d. KO, knockout. (B) Spleen and liver of wild-type (WT; n = 7), IFN-γ–deficient (n = 7), and LRG-47–deficient mice (n = 9) were isolated at 3 d after infection, and the numbers of bacteria present was determined. Statistical analysis for wild-type vs. LRG-47–deficient mice: spleen, P = 0.003; liver, P = 0.05. (C) Sera were isolated from wild-type (n = 3) and LRG-47–deficient mice (n = 5) at 3 d after infection and used for IFN-γ and IL-12 determination by ELISA. A–C are representative results from two experiments.

Figure 4

Figure 4

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.

Figure 6

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) Spleen and liver of wild-type (n = 4), IFN-γ–deficient (n = 3), and IRG-47–deficient mice (n = 5) were isolated at 3 d after infection, and the number of bacteria present was determined. Statistical analysis for wild-type vs. IRG-47–deficient mice: spleen, P = 0.01; liver, P = 0.28. The slight increase in the splenic bacterial loads of IRG-47–deficient mice, compared with that of wild-type mice, was not seen in a second experiment. A and B are representative results from two experiments.

Figure 6

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) Spleen and liver of wild-type (n = 4), IFN-γ–deficient (n = 3), and IRG-47–deficient mice (n = 5) were isolated at 3 d after infection, and the number of bacteria present was determined. Statistical analysis for wild-type vs. IRG-47–deficient mice: spleen, P = 0.01; liver, P = 0.28. The slight increase in the splenic bacterial loads of IRG-47–deficient mice, compared with that of wild-type mice, was not seen in a second experiment. A and B are representative results from two experiments.

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References

    1. Stark G.R., Kerr I.M., Williams B.R.G., Silverman R.H., Shreiber R.D. How cells respond to interferons. Annu. Rev. Biochem. 1998;67:227–264. - PubMed
    1. Boehm U., Klamp T., Groot M., Howard J.C. Cellular responses to interferon-γ. Annu. Rev. Immunol. 1997;15:749–795. - PubMed
    1. Der S.D., Zhou A., Williams B.R.G., Silverman R.H. Identification of genes differetially regulated by interferon α, β, or γ using oligonucleotide arrays. Proc. Natl. Acad. Sci. USA. 1998;95:15623–15628. - PMC - PubMed
    1. Sorace J.M., Johnson R.J., Howard D.L., Drysdale B.E. Identification of an endotoxin and IFN-γ-inducible cDNApossible identification of a novel protein family. J. Leukoc. Biol. 1995;58:477–484. - PubMed
    1. Taylor G.A., Stauber R., Rulong S., Hudson S.E., Pei V., Pavlakis G.N., Resau J.H., Vande Woude G.F. The inducibly expressed GTPase (IGTP) localizes to the endoplasmic reticulum independently of GTP binding. J. Biol. Chem. 1997;272:10639–10645. - PubMed

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