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Loss of the interferon-γ-inducible regulatory immunity-related GTPase (IRG), Irgm1, causes activation of effector IRG proteins on lysosomes, damaging lysosomal function and predicting the dramatic susceptibility of Irgm1-deficient mice to infection.

Maric-Biresev J, Hunn JP, Krut O, Helms JB, Martens S, Howard JC - BMC Biol. (2016)

Bottom Line: We show that the three regulatory IRG proteins (GMS sub-family), including Irgm1, each of which localizes to distinct sets of endocellular membranes, play an important role during the cellular response to IFN-γ, each protecting specific membranes from off-target activation of effector IRG proteins (GKS sub-family).In the absence of Irgm1, which is localized mainly at lysosomal and Golgi membranes, activated GKS proteins load onto lysosomes, and are associated with reduced lysosomal acidity and failure to process autophagosomes.The membrane targeting properties of the three GMS proteins to specific endocellular membranes prevent accumulation of activated GKS protein effectors on the corresponding membranes and thus enable GKS proteins to distinguish organellar cellular membranes from the membranes of pathogen vacuoles.

View Article: PubMed Central - PubMed

Affiliation: Institute for Genetics, University of Cologne, Cologne, Germany.

ABSTRACT

Background: The interferon-γ (IFN-γ)-inducible immunity-related GTPase (IRG), Irgm1, plays an essential role in restraining activation of the IRG pathogen resistance system. However, the loss of Irgm1 in mice also causes a dramatic but unexplained susceptibility phenotype upon infection with a variety of pathogens, including many not normally controlled by the IRG system. This phenotype is associated with lymphopenia, hemopoietic collapse, and death of the mouse.

Results: We show that the three regulatory IRG proteins (GMS sub-family), including Irgm1, each of which localizes to distinct sets of endocellular membranes, play an important role during the cellular response to IFN-γ, each protecting specific membranes from off-target activation of effector IRG proteins (GKS sub-family). In the absence of Irgm1, which is localized mainly at lysosomal and Golgi membranes, activated GKS proteins load onto lysosomes, and are associated with reduced lysosomal acidity and failure to process autophagosomes. Another GMS protein, Irgm3, is localized to endoplasmic reticulum (ER) membranes; in the Irgm3-deficient mouse, activated GKS proteins are found at the ER. The Irgm3-deficient mouse does not show the drastic phenotype of the Irgm1 mouse. In the Irgm1/Irgm3 double knock-out mouse, activated GKS proteins associate with lipid droplets, but not with lysosomes, and the Irgm1/Irgm3(-/-) does not have the generalized immunodeficiency phenotype expected from its Irgm1 deficiency.

Conclusions: The membrane targeting properties of the three GMS proteins to specific endocellular membranes prevent accumulation of activated GKS protein effectors on the corresponding membranes and thus enable GKS proteins to distinguish organellar cellular membranes from the membranes of pathogen vacuoles. Our data suggest that the generalized lymphomyeloid collapse that occurs in Irgm1(-/-) mice upon infection with a variety of pathogens may be due to lysosomal damage caused by off-target activation of GKS proteins on lysosomal membranes and consequent failure of autophagosomal processing.

No MeSH data available.


Related in: MedlinePlus

Irgm1−/− mouse embryonic fibroblasts (MEFs) and bone marrow derived macrophages (BMDMs) do not die upon IFN-γ induction. a Wild type (WT) and Irgm1−/− MEFs were induced with 200 U/mL IFN-γ or treated with 10 mM LeuLeuOMe for 24, 48, and 72 hours, or left untreated. Cells were stained with nuclear dyes Hoechst (all cells) and propidium iodide (PI; dead cells only). Representative microscopic images of Hoechst- and PI-stained WT cells are shown. Scale bar represents 100 μM. b Quantification of 8a showing the percentage of PI-positive MEFs. Dye stained nuclei were quantified with the Volocity software; 10 pictures (5000–10000 cells) per sample were quantified and the means of three independent experiments ± standard deviation are shown. c WT and Irgm1−/− BMDM cells were induced with 200 U/mL IFN-γ, 500 ng/mL lipopolysaccharide, 5 mM LeuLeuOMe for 24, 48, and 72 hours, or left untreated. The experiment was performed as in 8a, b; 10 pictures (5000–10000 cells) per sample were quantified and the means of three independent experiments ± standard deviation are shown
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Fig8: Irgm1−/− mouse embryonic fibroblasts (MEFs) and bone marrow derived macrophages (BMDMs) do not die upon IFN-γ induction. a Wild type (WT) and Irgm1−/− MEFs were induced with 200 U/mL IFN-γ or treated with 10 mM LeuLeuOMe for 24, 48, and 72 hours, or left untreated. Cells were stained with nuclear dyes Hoechst (all cells) and propidium iodide (PI; dead cells only). Representative microscopic images of Hoechst- and PI-stained WT cells are shown. Scale bar represents 100 μM. b Quantification of 8a showing the percentage of PI-positive MEFs. Dye stained nuclei were quantified with the Volocity software; 10 pictures (5000–10000 cells) per sample were quantified and the means of three independent experiments ± standard deviation are shown. c WT and Irgm1−/− BMDM cells were induced with 200 U/mL IFN-γ, 500 ng/mL lipopolysaccharide, 5 mM LeuLeuOMe for 24, 48, and 72 hours, or left untreated. The experiment was performed as in 8a, b; 10 pictures (5000–10000 cells) per sample were quantified and the means of three independent experiments ± standard deviation are shown

Mentions: To analyze the possibility of necrotic cell death, WT and Irgm1−/− MEFs were induced with IFN-γ or left untreated. Treatment with the LMP inducer LeuLeuOMe [54, 55] was used as a positive cell death control. After 24, 48, or 72 hours, live cells were stained with cell-permeant DNA binding Hoechst dye, which stains the nuclei of live and dead nuclei, and cell-impermeant propidium iodide (PI), which stains only the nuclei of dead cells (Fig. 8a). No difference in cell death between WT and Irgm1−/− MEFs was observed (Fig. 8b). We repeated this experiment in WT bone marrow derived macrophages (BMDM) and Irgm1−/− BMDMs, which were induced with IFN-γ, lipopolysaccharide, LeuLeuOMe, or left untreated (Fig. 8c). As previously reported in a similar analysis [6], no difference in cell death between the two cell populations was observed.Fig. 8


Loss of the interferon-γ-inducible regulatory immunity-related GTPase (IRG), Irgm1, causes activation of effector IRG proteins on lysosomes, damaging lysosomal function and predicting the dramatic susceptibility of Irgm1-deficient mice to infection.

Maric-Biresev J, Hunn JP, Krut O, Helms JB, Martens S, Howard JC - BMC Biol. (2016)

Irgm1−/− mouse embryonic fibroblasts (MEFs) and bone marrow derived macrophages (BMDMs) do not die upon IFN-γ induction. a Wild type (WT) and Irgm1−/− MEFs were induced with 200 U/mL IFN-γ or treated with 10 mM LeuLeuOMe for 24, 48, and 72 hours, or left untreated. Cells were stained with nuclear dyes Hoechst (all cells) and propidium iodide (PI; dead cells only). Representative microscopic images of Hoechst- and PI-stained WT cells are shown. Scale bar represents 100 μM. b Quantification of 8a showing the percentage of PI-positive MEFs. Dye stained nuclei were quantified with the Volocity software; 10 pictures (5000–10000 cells) per sample were quantified and the means of three independent experiments ± standard deviation are shown. c WT and Irgm1−/− BMDM cells were induced with 200 U/mL IFN-γ, 500 ng/mL lipopolysaccharide, 5 mM LeuLeuOMe for 24, 48, and 72 hours, or left untreated. The experiment was performed as in 8a, b; 10 pictures (5000–10000 cells) per sample were quantified and the means of three independent experiments ± standard deviation are shown
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Fig8: Irgm1−/− mouse embryonic fibroblasts (MEFs) and bone marrow derived macrophages (BMDMs) do not die upon IFN-γ induction. a Wild type (WT) and Irgm1−/− MEFs were induced with 200 U/mL IFN-γ or treated with 10 mM LeuLeuOMe for 24, 48, and 72 hours, or left untreated. Cells were stained with nuclear dyes Hoechst (all cells) and propidium iodide (PI; dead cells only). Representative microscopic images of Hoechst- and PI-stained WT cells are shown. Scale bar represents 100 μM. b Quantification of 8a showing the percentage of PI-positive MEFs. Dye stained nuclei were quantified with the Volocity software; 10 pictures (5000–10000 cells) per sample were quantified and the means of three independent experiments ± standard deviation are shown. c WT and Irgm1−/− BMDM cells were induced with 200 U/mL IFN-γ, 500 ng/mL lipopolysaccharide, 5 mM LeuLeuOMe for 24, 48, and 72 hours, or left untreated. The experiment was performed as in 8a, b; 10 pictures (5000–10000 cells) per sample were quantified and the means of three independent experiments ± standard deviation are shown
Mentions: To analyze the possibility of necrotic cell death, WT and Irgm1−/− MEFs were induced with IFN-γ or left untreated. Treatment with the LMP inducer LeuLeuOMe [54, 55] was used as a positive cell death control. After 24, 48, or 72 hours, live cells were stained with cell-permeant DNA binding Hoechst dye, which stains the nuclei of live and dead nuclei, and cell-impermeant propidium iodide (PI), which stains only the nuclei of dead cells (Fig. 8a). No difference in cell death between WT and Irgm1−/− MEFs was observed (Fig. 8b). We repeated this experiment in WT bone marrow derived macrophages (BMDM) and Irgm1−/− BMDMs, which were induced with IFN-γ, lipopolysaccharide, LeuLeuOMe, or left untreated (Fig. 8c). As previously reported in a similar analysis [6], no difference in cell death between the two cell populations was observed.Fig. 8

Bottom Line: We show that the three regulatory IRG proteins (GMS sub-family), including Irgm1, each of which localizes to distinct sets of endocellular membranes, play an important role during the cellular response to IFN-γ, each protecting specific membranes from off-target activation of effector IRG proteins (GKS sub-family).In the absence of Irgm1, which is localized mainly at lysosomal and Golgi membranes, activated GKS proteins load onto lysosomes, and are associated with reduced lysosomal acidity and failure to process autophagosomes.The membrane targeting properties of the three GMS proteins to specific endocellular membranes prevent accumulation of activated GKS protein effectors on the corresponding membranes and thus enable GKS proteins to distinguish organellar cellular membranes from the membranes of pathogen vacuoles.

View Article: PubMed Central - PubMed

Affiliation: Institute for Genetics, University of Cologne, Cologne, Germany.

ABSTRACT

Background: The interferon-γ (IFN-γ)-inducible immunity-related GTPase (IRG), Irgm1, plays an essential role in restraining activation of the IRG pathogen resistance system. However, the loss of Irgm1 in mice also causes a dramatic but unexplained susceptibility phenotype upon infection with a variety of pathogens, including many not normally controlled by the IRG system. This phenotype is associated with lymphopenia, hemopoietic collapse, and death of the mouse.

Results: We show that the three regulatory IRG proteins (GMS sub-family), including Irgm1, each of which localizes to distinct sets of endocellular membranes, play an important role during the cellular response to IFN-γ, each protecting specific membranes from off-target activation of effector IRG proteins (GKS sub-family). In the absence of Irgm1, which is localized mainly at lysosomal and Golgi membranes, activated GKS proteins load onto lysosomes, and are associated with reduced lysosomal acidity and failure to process autophagosomes. Another GMS protein, Irgm3, is localized to endoplasmic reticulum (ER) membranes; in the Irgm3-deficient mouse, activated GKS proteins are found at the ER. The Irgm3-deficient mouse does not show the drastic phenotype of the Irgm1 mouse. In the Irgm1/Irgm3 double knock-out mouse, activated GKS proteins associate with lipid droplets, but not with lysosomes, and the Irgm1/Irgm3(-/-) does not have the generalized immunodeficiency phenotype expected from its Irgm1 deficiency.

Conclusions: The membrane targeting properties of the three GMS proteins to specific endocellular membranes prevent accumulation of activated GKS protein effectors on the corresponding membranes and thus enable GKS proteins to distinguish organellar cellular membranes from the membranes of pathogen vacuoles. Our data suggest that the generalized lymphomyeloid collapse that occurs in Irgm1(-/-) mice upon infection with a variety of pathogens may be due to lysosomal damage caused by off-target activation of GKS proteins on lysosomal membranes and consequent failure of autophagosomal processing.

No MeSH data available.


Related in: MedlinePlus