IFN-γ mediates Paneth cell death via suppression of mTOR
- Center for Vaccine Biology and Immunology, Department of Microbiology and Immunology, University of Rochester Medical Center, United States
- Department of Medicine and Department of Biomedical Sciences, Cedars-Sinai Medical Center, United States
- Cancer Prevention and Control Program, Fox Chase Cancer Center, United States
Figures
Figure 1 with 1 supplement
IFN-γ directly contributes to Paneth cell loss during Toxoplasma gondii infection.
(a) qRT-PCR analysis of relative Lyz1, Defa23, and Defa24 expression measured in the small intestines of WT, E-Ifngr2 KO, and PC- Ifngr2 KO infected orally with 20 cysts per mouse of the ME49 T. …
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Figure 1—source data 1
Analysis of Paneth cells in the small intestines of WT, E-Ifngr2 KO, and PC- Ifngr2 KO infected orally with Toxoplasma gondii.
Figure 1A: qRT-PCR analysis of relative Lyz1, Defa23, and Defa24 expression measured in the small intestines of WT, E-Ifngr2 KO, and PC- Ifngr2 KO infected orally with 20 cysts per mouse of the ME49 T. gondii on day 7 after infection. Figure 1C: Quantification of Paneth cells per crypt in naïve and infected WT, E- Ifngr2 KO, and PC- Ifngr2 KO mice with 20 cysts of ME49 T. gondii on day 7. Figure 1F: numbers of PI+ puncta per crypt in the small intestines of WT, E-Ifngr2 KO, and PC- Ifngr2 KO infected orally with 20 cysts per mouse of the ME49 T. gondii on day 7 after infection.
- https://cdn.elifesciences.org/articles/60478/elife-60478-fig1-data1-v2.xlsx
Figure 1—figure supplement 1
Visualization of Paneth cell death during Toxoplasma gondii infection in vivo.
(a) Immunofluorescence images of mouse small intestine on day 6 post infection with T. gondii stained in vivo with propidium iodide (PI, red). Paneth cells were detected with anti-Lyz1 staining …
Figure 2 with 2 supplements
IFN-γ triggers Paneth cells death independently of apoptosis.
Immunofluorescence images of crypts of WT mice on days 3, 5, and 7 post Toxoplasma gondii infection. Images were stained with UEA (green), DAPI (blue), and (a) Cleaved Caspase 3 (pink) or (b) TUNEL. …
Figure 2—figure supplement 1
IFN-γ triggers Paneth cells death independently of apoptosis in vitro.
(a) Intestinal organoids were generated from small intestinal stem cells isolated from crypts of PC-Cre × TdTomato reporter mice and stimulated with IFN-γ (200 ng/ml) alone or in the presence of …
Figure 2—figure supplement 2
IFN-γ-mediated Paneth cell loss occurs independently of pyroptosis or necroptosis.
(a) Immunofluorescence images of intestinal crypts in naïve and Toxoplasma gondii-infected (day 7 post infection) WT, Casp1/11−/−, Nlrp3−/−, and Pycard−/− mice stained with UEA (green), Lyz1 (pink), …
Figure 3 with 1 supplement
Toxoplasma gondii infection leads to IFN-γ-mediated inhibition of mTORC1 activity in Paneth cells.
(a) Immunofluorescence images of small intestinal crypts from naïve, T. gondii-infected and T. gondii-infected mice treated with anti-IFN-γantibody on days 3, 5, and 7 post oral infection with T. …
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Figure 3—source data 1
Toxoplasma gondii infection leads to IFN-γ-mediated inhibition of mTORC1 activity in Paneth cells.
Figure 3B qRT-PCR analysis for the relative Lyz1 expression in small intestines of naïve and T. gondii-infected mice on days 3, 5, and 7 post infection. Figure 3C: qRT-PCR analysis for the relative IFN-γ and Irgm1 expression in small intestines of naïve and T. gondii-infected mice on days 3, 5, and 7 post infection. Figure 3E Mean fluorescent intensity of p-Ser 240/244 channel in individual intestinal sections of T. gondii-infected WT, E-ifngr2 KO, and PC-ifngr2 KO mice on day 7 post infection.
- https://cdn.elifesciences.org/articles/60478/elife-60478-fig3-data1-v2.xlsx
Figure 3—figure supplement 1
mTOR activity in Paneth cells and intestinal stem cells in vivo.
(a) Confocal immunofluorescence images of intestinal crypts prepared from the PC-Cre x TdTomato x Lgr5-GFP double reporter mice. Stem cells are green (Lgr5+), Paneth cells are red (TdTomato+) and …
Figure 4
IFN-γ is sufficient for inhibition of mTORC1 activity in Paneth cells.
(a) Mice were treated with recombinant IFN-γ for 24 hr (D1), 48 hr (D2), and 72 hr (D3), and the serum levels of the cytokine were analyzed by ELISA in comparison to Toxoplasma gondii-infected mice …
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Figure 4—source data 1
IFN-γ is sufficient for inhibition of mTORC1 activity in Paneth cells.
Figure 4A: the serum levels of IFN-γ were analyzed by ELISA in comparison to Toxoplasma gondii-infected mice on day 7 post infection. Figure 4B qRT-PCR analysis for the relative Defa24 and Lyz1 expression in small intestines of mice treated with recombinant IFN-γ. Figure 4F: The frequency and absolute numbers of mTOR+ Paneth cells in control and IFN-γ treated mice.
- https://cdn.elifesciences.org/articles/60478/elife-60478-fig4-data1-v2.xlsx
Figure 5 with 1 supplement
Paneth cells require intrinsic mTORC1 signaling.
(a) Immunofluorescence images of small intestinal crypts of WT mice and mice lacking the mTOR regulatory protein Raptor (PC-Raptor) or mTOR kinase (PC-Mtor) in Paneth cells that were detected by UEA …
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Figure 5—source data 1
Paneth cells require intrinsic mTORC1 signaling.
Figure 5B qRT-PCR analysis of Lyz1, Defa23, and Defa24 relative expression in the small intestines of WT mice and mice lacking Raptor or mTOR in Paneth cells. Figure 5D qRT-PCR analysis for relative Lyz1, Defa23, and Defa24 expression in the small intestines in WT mice treated with rapamycin daily for 24 hr (D1) or 72 hr (D3). WT, wild-type.
- https://cdn.elifesciences.org/articles/60478/elife-60478-fig5-data1-v2.xlsx
Figure 5—figure supplement 1
Paneth cell mTORC1 dependency is downstream of Toxoplasma gondii-infection triggered IFN-γ.
(a) Confocal immunofluorescence images of intestinal crypts prepared from naïve or T. gondii-infected mice treated intraperitoneally with rapamycin (rapa). Sections were stained with UEA (red), …
Figure 6
IFN-γ-mediated mitochondrial damage accompanies mTORC1 inhibition in Paneth cells.
(a, b) Flow cytometric analysis of Paneth cell mitochondrial activity (potentiation) and mass analyzed by mitostatus and mitotracker staining, respectively, in naïve or Toxoplasma gondii-infected …
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Figure 6—source data 1
IFN-γ-mediated mitochondrial damage accompanies mTORC1 inhibition in Paneth cells.
Figure 6B Flow cytometric quantification of mitostatus and mitotracker positive Paneth cells in naïve or Toxoplasma gondii-infected PC-Cre x Rosa-YFP reporter mice on day 5 post infection. Figure 6E: MitoStatus MFI of the individual intestinal organoids treated with IFN-γ in vitro for 24 hr. Figure 6F: qRT-PCR analysis for relative Defa23 expression in organoids stimulated with IFN-γ (200 ng/ml) for 24 hr. Figure 6H: qRT-PCR analysis for relative Defa23 and Olfm4 expression in PC-Cre x TdTomato organoids stimulated with IFN-γ(200 ng/ml), Rapamycin (500 nM) or FCCP (100 µM) for 6 hr. MFI, mean fluorescence intensity.
- https://cdn.elifesciences.org/articles/60478/elife-60478-fig6-data1-v2.xlsx
Figure 7 with 1 supplement
Microbiota is required for IFN-γ-mediated mTORC1 inhibition during Toxoplasma gondii infection.
(a) qRT-PCR analysis for relative expression of Lyz1, Defa23, and Defa24 measured in the small intestines of conventional (CONV) and germ-free (GF) C57Bl/6 mice infected orally with 20 cysts of the …
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Figure 7—source data 1
Microbiota is required for IFN-γ-mediated mTORC1 inhibition during Toxoplasma gondii infection.
Figure 7A: qRT-PCR analysis for relative expression of Lyz1, Defa23, and Defa24 measured in the small intestines of conventional (CONV) and germ-free (GF) C57Bl/6 mice infected orally with 20 cysts of the ME49 T. gondii (day 7 post infection). Figure 7C: MFI quantification of p-Ser240/244 channel in individual intestinal sections of T. gondii-infected WT CONV and GF mice on day 7 post infection. MFI, mean fluorescence intensity; WT, wild-type.
- https://cdn.elifesciences.org/articles/60478/elife-60478-fig7-data1-v2.xlsx
Figure 7—figure supplement 1
Microbiota contributes to IFN-γ response during Toxoplasma gondii infection.
(a) qRT-PCR analysis for relative expression of IFN-γ expression in the small intestines of WT CONV and GF mice infected orally with 20 cysts of the ME49 T. gondii on day 7 post infection. Error …
Author response image 1
Western blot analysis of sort-sort-purified Paneth cells isolated from naïve or T. gondii infected mice (day 6 post infection).
Paneth cells were combined from 3 naïve mice (I) and 12-15 T. gondii infected mice (T). The data shown are from two independent experiments.
