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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

In the absence of Irgm1, Irga6 co-localizes with lysosomes. a Wild type (WT), Irgm1−/−, Irgm3−/−, and Irgm1/Irgm3−/− mouse embryonic fibroblasts (MEFs) were induced with 200 U/mL IFN-γ for 24 hours. Cells were fixed and stained with anti-Irga6 antiserum (165/3) and anti-LAMP1 antibody. Representative microscopic images of Irga6 and lysosome co-localization are shown. Arrows point at the Irga6 structures magnified at the end of each panel in the following array: upper left: Irga6, upper right: LAMP1, lower left: merge, lower right: phase contrast. Scale bars represent 10 μM. b Quantification of 2a, showing percentage of Irga6 aggregate-like structures co-localizing with LAMP1. Irga6 does not form aggregate-like structures in WT cells and therefore it was not quantified; 100 cells per sample were quantified and the means of three independent experiments ± standard deviation are shown. c Gene switch (gs) 3T3 cells stably transfected with inducible Irga6 were stimulated with mifepristone and simultaneously transiently transfected with pGW1H-Irgm1, pGW1H-Irgm2, and pGW1H-Irgm3 either alone or in combination and incubated for 24 hours. Samples were fixed and stained as in 1a. Representative images of Irga6 and lysosome co-localization are shown. Images of the cells transfected with additional combinations of GMS proteins are also included (Additional file 2: Figure S2). Arrows point at Irga6 structures magnified at the end of each panel in the following array: upper left: Irga6, upper right: LAMP1, lower left: overlay, lower right: phase contrast. Scale bars represent 10 μm. d Quantification of 2c and S2, showing percent of Irga6 structures co-localizing with LAMP1; 50 cells per sample were quantified and counts of two independent experiments are shown. eIrgm1−/− MEFs were induced with 200 U/mL IFN-γ and simultaneously transiently transfected with pEGFP-Irga6-ctag. Upon 24 h cells were fixed and stained for LAMP1. Scale bars represent 10 μM
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Fig2: In the absence of Irgm1, Irga6 co-localizes with lysosomes. a Wild type (WT), Irgm1−/−, Irgm3−/−, and Irgm1/Irgm3−/− mouse embryonic fibroblasts (MEFs) were induced with 200 U/mL IFN-γ for 24 hours. Cells were fixed and stained with anti-Irga6 antiserum (165/3) and anti-LAMP1 antibody. Representative microscopic images of Irga6 and lysosome co-localization are shown. Arrows point at the Irga6 structures magnified at the end of each panel in the following array: upper left: Irga6, upper right: LAMP1, lower left: merge, lower right: phase contrast. Scale bars represent 10 μM. b Quantification of 2a, showing percentage of Irga6 aggregate-like structures co-localizing with LAMP1. Irga6 does not form aggregate-like structures in WT cells and therefore it was not quantified; 100 cells per sample were quantified and the means of three independent experiments ± standard deviation are shown. c Gene switch (gs) 3T3 cells stably transfected with inducible Irga6 were stimulated with mifepristone and simultaneously transiently transfected with pGW1H-Irgm1, pGW1H-Irgm2, and pGW1H-Irgm3 either alone or in combination and incubated for 24 hours. Samples were fixed and stained as in 1a. Representative images of Irga6 and lysosome co-localization are shown. Images of the cells transfected with additional combinations of GMS proteins are also included (Additional file 2: Figure S2). Arrows point at Irga6 structures magnified at the end of each panel in the following array: upper left: Irga6, upper right: LAMP1, lower left: overlay, lower right: phase contrast. Scale bars represent 10 μm. d Quantification of 2c and S2, showing percent of Irga6 structures co-localizing with LAMP1; 50 cells per sample were quantified and counts of two independent experiments are shown. eIrgm1−/− MEFs were induced with 200 U/mL IFN-γ and simultaneously transiently transfected with pEGFP-Irga6-ctag. Upon 24 h cells were fixed and stained for LAMP1. Scale bars represent 10 μM

Mentions: To test this idea, immortalized WT mouse embryonic fibroblasts (MEFs), Irgm1−/− MEFs, Irgm3−/− MEFs, and Irgm1/Irgm3−/− MEFs, all on a C57BL/6 background, were induced with IFN-γ for 24 hours and stained for Irga6 and for the late endosome/lysosome marker lysosomal-associated membrane protein 1 (LAMP1). In IFN-γ-induced WT MEFs, Irga6 retained the typical smooth non-aggregated pattern associated with the inactive state [17]. Irga6 formed aggregates in all GMS mutant cells, but clear ring-like forms co-localized with LAMP1 only in Irgm1−/− cells (Fig. 2a, b). However, in Irgm1/Irgm3−/− cells, in which both lysosomes and ER should lack GMS proteins, Irga6 aggregates do not co-localize with either of these cytoplasmic structures but co-localize with lipid droplets, confirming a recent report [21] (Fig. 2a). The Irga6 accumulated at lysosomes in Irgm1-deficient cells was shown to be in the activated state by intense staining with the monoclonal antibody 10D7 (Additional file 1: Figure S1), shown previously to bind preferentially to the active state of Irga6 [33].Fig. 2


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)

In the absence of Irgm1, Irga6 co-localizes with lysosomes. a Wild type (WT), Irgm1−/−, Irgm3−/−, and Irgm1/Irgm3−/− mouse embryonic fibroblasts (MEFs) were induced with 200 U/mL IFN-γ for 24 hours. Cells were fixed and stained with anti-Irga6 antiserum (165/3) and anti-LAMP1 antibody. Representative microscopic images of Irga6 and lysosome co-localization are shown. Arrows point at the Irga6 structures magnified at the end of each panel in the following array: upper left: Irga6, upper right: LAMP1, lower left: merge, lower right: phase contrast. Scale bars represent 10 μM. b Quantification of 2a, showing percentage of Irga6 aggregate-like structures co-localizing with LAMP1. Irga6 does not form aggregate-like structures in WT cells and therefore it was not quantified; 100 cells per sample were quantified and the means of three independent experiments ± standard deviation are shown. c Gene switch (gs) 3T3 cells stably transfected with inducible Irga6 were stimulated with mifepristone and simultaneously transiently transfected with pGW1H-Irgm1, pGW1H-Irgm2, and pGW1H-Irgm3 either alone or in combination and incubated for 24 hours. Samples were fixed and stained as in 1a. Representative images of Irga6 and lysosome co-localization are shown. Images of the cells transfected with additional combinations of GMS proteins are also included (Additional file 2: Figure S2). Arrows point at Irga6 structures magnified at the end of each panel in the following array: upper left: Irga6, upper right: LAMP1, lower left: overlay, lower right: phase contrast. Scale bars represent 10 μm. d Quantification of 2c and S2, showing percent of Irga6 structures co-localizing with LAMP1; 50 cells per sample were quantified and counts of two independent experiments are shown. eIrgm1−/− MEFs were induced with 200 U/mL IFN-γ and simultaneously transiently transfected with pEGFP-Irga6-ctag. Upon 24 h cells were fixed and stained for LAMP1. Scale bars represent 10 μM
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Fig2: In the absence of Irgm1, Irga6 co-localizes with lysosomes. a Wild type (WT), Irgm1−/−, Irgm3−/−, and Irgm1/Irgm3−/− mouse embryonic fibroblasts (MEFs) were induced with 200 U/mL IFN-γ for 24 hours. Cells were fixed and stained with anti-Irga6 antiserum (165/3) and anti-LAMP1 antibody. Representative microscopic images of Irga6 and lysosome co-localization are shown. Arrows point at the Irga6 structures magnified at the end of each panel in the following array: upper left: Irga6, upper right: LAMP1, lower left: merge, lower right: phase contrast. Scale bars represent 10 μM. b Quantification of 2a, showing percentage of Irga6 aggregate-like structures co-localizing with LAMP1. Irga6 does not form aggregate-like structures in WT cells and therefore it was not quantified; 100 cells per sample were quantified and the means of three independent experiments ± standard deviation are shown. c Gene switch (gs) 3T3 cells stably transfected with inducible Irga6 were stimulated with mifepristone and simultaneously transiently transfected with pGW1H-Irgm1, pGW1H-Irgm2, and pGW1H-Irgm3 either alone or in combination and incubated for 24 hours. Samples were fixed and stained as in 1a. Representative images of Irga6 and lysosome co-localization are shown. Images of the cells transfected with additional combinations of GMS proteins are also included (Additional file 2: Figure S2). Arrows point at Irga6 structures magnified at the end of each panel in the following array: upper left: Irga6, upper right: LAMP1, lower left: overlay, lower right: phase contrast. Scale bars represent 10 μm. d Quantification of 2c and S2, showing percent of Irga6 structures co-localizing with LAMP1; 50 cells per sample were quantified and counts of two independent experiments are shown. eIrgm1−/− MEFs were induced with 200 U/mL IFN-γ and simultaneously transiently transfected with pEGFP-Irga6-ctag. Upon 24 h cells were fixed and stained for LAMP1. Scale bars represent 10 μM
Mentions: To test this idea, immortalized WT mouse embryonic fibroblasts (MEFs), Irgm1−/− MEFs, Irgm3−/− MEFs, and Irgm1/Irgm3−/− MEFs, all on a C57BL/6 background, were induced with IFN-γ for 24 hours and stained for Irga6 and for the late endosome/lysosome marker lysosomal-associated membrane protein 1 (LAMP1). In IFN-γ-induced WT MEFs, Irga6 retained the typical smooth non-aggregated pattern associated with the inactive state [17]. Irga6 formed aggregates in all GMS mutant cells, but clear ring-like forms co-localized with LAMP1 only in Irgm1−/− cells (Fig. 2a, b). However, in Irgm1/Irgm3−/− cells, in which both lysosomes and ER should lack GMS proteins, Irga6 aggregates do not co-localize with either of these cytoplasmic structures but co-localize with lipid droplets, confirming a recent report [21] (Fig. 2a). The Irga6 accumulated at lysosomes in Irgm1-deficient cells was shown to be in the activated state by intense staining with the monoclonal antibody 10D7 (Additional file 1: Figure S1), shown previously to bind preferentially to the active state of Irga6 [33].Fig. 2

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