EMBO/soda-nel huggingface dataset:
Response to interferons and antibacterial innate immunity in absence of tyrosine-phosphorylated STAT1 (2016)
Figure 1

Figure 1 - Interferon signaling in mice bearing heterozygous Stat1Y701F mutation resembles that of human cells with heterozygous Stat1Y701C mutation.

A Western blot analysis STAT expression and phosphorylation. Bone marrow derived macrophages (BMDMs) of wild type (WT), Stat1Y701F/+ (WT/YF) or Stat1-/- (S1) mice were treated with 250 IU/mL of IFNβ or 5 ng/mL of IFNγ for 0.5, 6, 12 and 24 h. Whole cell extracts were collected and tested in western blot for levels of phosphorylation of STAT1 (Y701) and STAT2 (Y689) and total level of STAT1 and STAT2. The blots are representative of more than 3 independent experiments.

B Effect of STAT1Y701Fheterozygosity on the expression of type I IFN-induced genes (ISG). BMDMs of wild type (WT), Stat1Y701F/+ (WT/YF) or Stat1-/- (S1) mice were treated with 250 IU/mL of IFNβ for 4 and 48 h. Gene expression was measured by Q-PCR and normalized to Gapdh and to the expression levels in untreated wild type cells. The bars represent mean values with the standard deviations (SD) of three independent experiments.

C Effect of STAT1Y701Fheterozygosity on the expression of IFNγ-induced genes. BMDMs of wild type (WT), Stat1Y701F/+ (WT/YF) or Stat1-/- (S1) mice were treated with 5 ng/mL of IFNγ for 4 and 48 h. Gene expression was measured by Q-PCR and normalized to Gapdh and to the expression levels in untreated wild type cells. The bars represent mean values with the standard deviations (SD) of three independent experiments.
Response to interferons and antibacterial innate immunity in absence of tyrosine-phosphorylated STAT1 (2016)
Figure 2

Figure 2 - STAT1 expression and interferon signaling in Stat1Y701F mice.

A Effect of STAT1Y701F homozygosity on STAT1 expression in mouse cells and organs. Spleens, livers or bone marrow derived macrophages (BMDMs) were isolated from wild type (WT), Stat1Y701F (YF) and Stat1-/- (S1) mice. Whole cell extracts were collected and tested for levels of total Stat1 in western blot. The blots are representative of more than 3 independent experiments.

B Effect of STAT1Y701F homozygosity on STAT1 phosphorylation at Y701. BMDMs were isolated from wild type (WT), Stat1Y701F (YF) and Stat1-/- (S1) mice and stimulated for 30 min with 250 IU/mL of IFNβ or 5 ng/mL of IFNγ. Whole cell extracts were collected and tested for levels of STAT1 phosphorylation on Y701 in western blot. The blots are representative of more than 3 independent experiments.

C Effect of STAT1Y701Fhomozygosity on the expression of IFNγ-induced genes. BMDMs of wild type (WT), Stat1Y701F (YF) and Stat1-/- (S1) mice were treated with 5 ng/mL of IFNγ for 4 or 48 h. Gene expression was measured by Q-PCR and normalized to Gapdh and to the expression levels in untreated wild type cells. Bars represent a mean value of 3 independent experiments. Error bars represent standard error of the mean (SEM); asterisks denote the level of statistical significance (ns, p>0.05); the p-values were calculated using paired ratio t-test.

D Effect of STAT1Y701Fhomozygosity on the expression of type I IFN-induced genes (ISG). BMDMs were isolated from wild type (WT), Stat1Y701F, Stat1-/-, Stat2-/- and IRF9-/- mice treated with 250 IU/mL of IFNβ for 4, 8, 12, 24 or 48 h. Gene expression was measured by Q-PCR and normalized to Gapdh and to the expression levels in untreated wild type cells. Bars represent a mean value of 3 independent experiments. Error bars represent standard error of the mean (SEM); asterisks denote the level of statistical significance (ns, p>0.05; *, p≤ 0.05; **, p≤ 0.01); the p-values were calculated using paired ratio t-test.

E STAT1 and STAT2 phosphorylation in Stat1-/-, Stat1Y701F, Stat2-/- and Irf9-/- macrophages. BMDMs were isolated from wild type (WT), Stat1Y701F (YF), Stat1-/- (S1), Stat2-/- (S2) and IRF9-/- (IRF9) mice and treated with 250 IU/mL of IFNβ for 30 min or 6, 12 or 24 h. The whole cell extracts were collected and tested in western blot for levels of phosphorylation of STAT1 on Y701 and of STAT2 on Y689. The same cell extracts were tested for total levels of STAT1 and STAT2. The blots are representative of more than 3 independent experiments.
Response to interferons and antibacterial innate immunity in absence of tyrosine-phosphorylated STAT1 (2016)
Figure 3

Figure 3 - Presence of the STAT1Y701F mutant reduces IFNβ-stimulated binding of STAT2 to nuclear ISRE sequences.

A IFNβ-stimulated binding of STAT2 to ISRE sequences of Mx2 and IRF7 promoters. Bone marrow derived macrophages (BMDMs) of wild type (WT), Stat1Y701F (YF), Stat1-/- (S1), Stat2-/- (S2) and IRF9-/- (IRF9) mice were treated with 250 IU/mL of IFNβ for 2 or 24 h. Cells were crosslinked, sonicated and immunoprecipitated with STAT2-specific antibody. The amount of precipitated DNA was measured by Q-PCR. Bars represent a mean value of 3 independent experiments. Error bars represent standard error of the mean (SEM); asterisks denote the level of statistical significance (**, p≤ 0.01); the p-values were calculated using paired ratio t-test.

B Impact of STAT2 deficiency on IFNβ-stimulated STAT1 association with the Mx2 ISRE. BMDMs of wild type (WT), Stat1-/- (S1) and Stat2-/- (S2) mice were treated with 250 IU/mL of IFNβ for 2 or 24 h. Cells were crosslinked, sonicated and immunoprecipitated with STAT1-specific antibody. The amount of precipitated DNA was measured by Q-PCR. Bars represent mean values of three independent experiments; error bars represent standard error of mean (SEM).

C Simultaneous association of STAT1 and STAT2 with the Mx2 ISRE analyzed by ChIP-reChIP. BMDMs of wild type (WT) mice were treated with 250 IU/mL of IFNβ for 2 or 24 h. Cells were crosslinked, sonicated and immunoprecipitated with either STAT1-specific antibody and re-immunoprecipitated with STAT2-specific antibody or vice versa. The amount of precipitated DNA was measured by Q-PCR. Bars represent mean values of three independent experiments; error bars represent standard deviation (SD).

D Impact of STAT1Y701F on delayed, STAT2-mediated expression of IFN-induced genes. Stat1-/- fibroblasts were transfected with plasmids driving expression of the indicated proteins. 24 h after transfection, 250 IU/ml of IFNβ was added to the transfected cells and ISG expression was determined by Q-PCR after 48h of cytokine treatment. Gene expression was measured by Q-PCR and normalized to Gapdh. Bars represent a mean value of 3 independent experiments. Error bars represent standard error of the mean (SEM) and asterisks denote the level of statistical significance (*, p≤ 0.05); the p-values were calculated using paired ratio t-test.
Response to interferons and antibacterial innate immunity in absence of tyrosine-phosphorylated STAT1 (2016)
Figure 4

Figure 4 - STAT1Y701F mutant reduces IFNβ-stimulated nuclear translocation of STAT2.

A Analysis of STAT2 nuclear translocation by immunofluorescence. Bone marrow derived macrophages (BMDMs) of wild type (WT), Stat1Y701F (YF), Stat1-/- (S1), Stat2-/- (S2) and IRF9-/- (IRF9) mice were seeded on cover slips and stimulated with 250 IU/mL of IFNβ for 30 min or 24 h. The cells were fixed and stained for STAT2 specific antibody followed by Alexafluor® 488 conjugated secondary antibody (green). Nuclei were stained with DAPI (blue). Studies are representative of more than three independent experiments. The scale bars represent 10 µm.

B Quantitative evaluation of STAT2 nuclear translocation. The intensity of STAT2-dependent immunofluorescence over DNA staining (DAPI) was quantified using ImageJ software in 20 cells from two independent experiments. Bars represent a mean with standard deviation (SD) and asterisks denote the level of statistical significance (***, p≤ 0.001); p-value was calculated using unpaired t-test.
Response to interferons and antibacterial innate immunity in absence of tyrosine-phosphorylated STAT1 (2016)
Figure 5

Figure 5 - STAT1Y701F mutant counteracts the inhibition of L. pneumophillareplication by delayed, STAT2/IRF9-dependent IFN signaling.

A Legionella pneumophila growth in unstimulated macrophages. Bone marrow derived macrophages (BMDMs) of wild type (WT), Stat1Y701F, Stat1-/- mice were seeded on in the 24-well plates and infected with L. pneumophila (JR32 Fla-, MOI 0.25). The numbers of colony forming units (CFUs) were determined 24 h, 48 h and 72 h after infection on charcoal yeast extract plates (CYE). The 0 time point was collected 1.5 h after the infection.

B Legionella pneumophila growth in IFNβ-treated macrophages. Bone marrow derived macrophages (BMDMs) of wild type (WT), Stat1Y701F, Stat1-/- mice were seeded in 24-well plates, treated with 500 U/mL of IFNβ and then infected with L. pneumophila (JR32 Fla-, MOI 0.25). The numbers of colony forming units (CFUs) were determined 24 h, 48 h and 72 h after infection on charcoal yeast extract plates (CYE). The 0 time point was collected 1.5 h after the infection.

The studies in A and B represent six biological repeats and the data are represented as mean values. Asterisks denote statistically significant differences between CFU numbers from Stat1Y701F and Stat1-/- cells (***, p≤ 0.001); p-values were calculated using unpaired t-test.
Response to interferons and antibacterial innate immunity in absence of tyrosine-phosphorylated STAT1 (2016)
Figure 6

Figure 6 - Interferon signaling in L. monocytogenes infected Stat1Y701F bone marrow derived macrophages.

A Western blot analysis of STAT1 tyrosine phosphorylation. Bone marrow derived macrophages (BMDMs) of wild type (WT), Stat1Y701F (YF) and Stat1-/- (S1) mice were infected with L. monocytogenes (LO28, MOI 10) for 5 or 6 h. Whole cell extracts were collected and tested in western blot for levels of total STAT1 and phosphorylation of STAT1 on tyrosine (Y701). The blots are representative of more than 3 independent experiments.

B Impact of Stat1Y701F mutation, or of deletion of ISGF3 subunits on the expression of the IFNβ gene. BMDMs of wild type (WT), Stat1Y701F, Stat1-/-, Stat2-/- and IRF9-/- mice were infected with L. monocytogenes (LO28, MOI 10) for 4, 8, 12, 24 or 48 h. Levels of Ifnβ gene expression were determined by Q-PCR. Bars represent mean values of three independent experiments. Error bars represent standard error of mean (SEM).
Response to interferons and antibacterial innate immunity in absence of tyrosine-phosphorylated STAT1 (2016)
Figure 7

Figure 7 - STAT1Y701F contributes to clearance of L. monocytogenes infection.

A Impact of Stat1 deficiency or of STAT1Y701F mutation on the growth of Listeria monocytogenes in macrophages. Bone marrow derived macrophages (BMDMs) of wild type (WT), Stat1Y701F and Stat1-/- mice were infected with L. monocytogenes (LO28, MOI 10). Colony forming unit (CFU) numbers were determined 1, 2, 4, 6 or 8 h after infection by plating on brain-heart-infusion (BHI) agar plates. The graph represents biological triplicates and the data are represented as mean values. Error bars represent standard deviation (SD) and asterisks denote statistically significant differences (ns, p>0.05; **, p≤0.01; ***, p≤ 0.001); p-values were calculated using unpaired t-test.

B Survival of mice infected with L. monocytogenes. 10 wild type (WT), Stat1Y701F and Stat1-/- mice per group were infected by intraperitoneal injection of 1x 102 viable L. monocytogenes. Survival was monitored over 10 days. The study is representative of more than 3 independent experiments.

C Organ pathogen burdens of mice infected with L. monocytogenes. Wild type (WT), Stat1Y701F (YF) and Stat1-/- (S1) mice were infected by intraperitoneal injection of 1x 102 viable L. monocytogenes. Number of colony forming units (CFU) in organs was determined at 48 h, 72 h or at the terminal stage of infection by plating homogenates on brain-heart-infusion (BHI) agar plates or Oxford agar plates (for lungs). Dots represent pooled data of 3 independent experiments. Lines represent the median and asterisks denote statistically significant differences (ns, p>0.05; *, p≤ 0.05; **, p≤ 0.01; ***, p≤ 0.001; ****, p≤ 0.0001); p-values were calculated using Mann-Whitney test.
Response to interferons and antibacterial innate immunity in absence of tyrosine-phosphorylated STAT1 (2016)
Figure 8

Figure 8 - Liver inflammation in Stat1Y701F and Stat1-/- mice after infection with Listeria monocytogenes.

A Immunohistochemical analysis of infection and inflammatory infiltrates. Wild type (WT), Stat1Y701F and Stat1-/- mice were infected by intraperitoneal injection of 1x102 viable L. monocytogenes for 48 h. Liver sections were examined by immunohistochemistry with L. monocytogenes or Ly6C/Ly6G specific antibody. The scale bars on 20x magnification images represent 100 µm and 50 µm on the 63x magnification images.

B Quantitative evaluation of inflammatory infiltrates. Infiltrates representing the entire surface of sections from five animals per genotype were counted and categorized according to their size.

C Liver pathology in infected mice. Wild type (WT), Stat1Y701F (YF) and Stat1-/- (S1) mice were infected by intraperitoneal injection of 1x105 viable L. monocytogenes for 72 h. Serum was collected and tested for ALT activity. Lines represent the median and asterisks denote statistically significant differences (*, p≤ 0.05; ***, p≤ 0.001); p-values were calculated using unpaired t-test.
Microglial phagocytosis of living photoreceptors contributes to inherited retinal degeneration. (2015)
Figure 1

Early translocation of retinal microglia from the inner to the outer nuclear layer (ONL) during rod degeneration in rd10 mice

A–D Progression of microglial infiltration in the ONL. Early in the course of rod degeneration at P18, few isolated apoptotic TUNEL-positive nuclei (red, inset) were present in the ONL, with Iba1+ microglial somata and processes (green) confined to the inner retina (A). At P21, microglia in the outer plexiform layer (OPL) extended radial processes into the ONL (inset), with a concurrent increase in the number of TUNEL+ nuclei (B). At P30, retinal microglia infiltrated the entire depth of the markedly thinned ONL and the subretinal space (SRS) (inset) (C). At P46, microglia became less dense in the ONL and reverted to a more ramified morphology (D). Scale bar, 50 μm.

E–G Quantitative analyses demonstrate that ONL atrophy progression, characterized by ONL thickness (empty symbols) and number of rows of ONL nuclei (filled symbols) (E), occurred concurrently with the progression of ONL apoptosis, followed as the density of TUNEL+ nuclei in the ONL (F), and microglia infiltration, followed as microglial density in the ONL (G) (n = 3 animals at each time point). Data points and error bars indicate mean ± SEM.

H–K Higher magnification images showing microglial process infiltration (H), phagosome formation in infiltrating processes (I), and microglial transformation into amoeboid morphologies containing multiple phagosomes (J,K).
Microglial phagocytosis of living photoreceptors contributes to inherited retinal degeneration. (2015)
Figure 2

Transient expression of phagocytic molecules and exposure of the “eat-me” signal phosphatidylserine (PS) in the outer nuclear layer during rod degeneration

A–C Microglia infiltrating the ONL demonstrate upregulation of the phagocytic molecule, CD68. Top images in (A) show composites of CD68 (red), Iba1 (green), and DAPI (blue) staining; bottom images show the same field with CD68 staining only. At P18, non-infiltrating microglia expressed low or undetectable levels of CD68, a lysosome-associated membrane protein. At P21–23, microglia infiltrating the ONL strongly upregulated CD68 (arrowheads). At P30, CD68 immunopositivity in ONL microglia decreased and was mostly confined to amoeboid cells in the subretinal space. (B) Magnified view of inset in (A) demonstrates localization of CD68 to microglia phagosomes. (C) CD68 expression (area of immunopositivity as a fraction of the ONL) demonstrated a peaked at P22 and decreased by P30. Scale bar, 20 μm.

D–F Upregulation of MFG-E8, a secreted glycoprotein that serves as a bridging molecule for phagocytosis receptors, in the ONL. (D) MFG-E8 was low or absent in the ONL at P18, but emerged at P21–23, localizing to photoreceptor cell bodies in a column-like pattern (arrow) and as a punctate signal within infiltrating microglia (arrowhead), before decreasing throughout the ONL at P30. (E) A magnified orthogonal view of the inset from (D) demonstrating punctate MFG-E8 immunopositivity within ONL microglia. (F) Quantification of MFG-E8 expression demonstrated a prominent emergence at P22 and a subsequent decrement by P30. Scale bar, 20 μm.

G–K Increase in phosphatidylserine (PS) exposure in the ONL during photoreceptor degeneration. (G) At P18, PS immunopositivity is near absent in the ONL, but increased significantly in ONL somata at P21–23, before decreasing at P30. (H) Quantitation of PS exposure by image analysis (by fractional area of PS immunopositivity within the ONL (top), and the mean intensity of PS staining in the ONL (bottom)) demonstrated a transient increase at P22. (I) Co-immunolabeling of rods with rhodopsin at P22 demonstrates that PS exposure was present in a majority of rods (inset shows at high magnification the co-labeling of PS and rhodopsin in multiple rod somata). (J) Conversely, immunolabeling of cones with cone arrestin demonstrates the sparse co-localization of PS in cones (inset shows close juxtaposition but no colocalization of PS and arrestin labeling). (K) Scoring of rhodopsin+ rods and arrestin+ cones for PS co-labeling demonstrates that a large majority of rods, but only a small minority of cones, showed PS exposure (two-sided unpaired t-test, n = 3 animals at P22). Scale bars = 20 μm.

Data information: Quantitative analyses in (C, F, H, and K) involved three animals at each time point. Column heights and error bars indicate mean ± SEM.
Microglial phagocytosis of living photoreceptors contributes to inherited retinal degeneration. (2015)
Figure 3

Infiltrating microglia phagocytose non-apoptotic photoreceptor rods during rod degeneration

A–C Phagocytosis of rods by infiltrating microglia. (A) Representative example of a Iba1+ microglial process extending into the ONL with a phagosome at its terminal end. Each phagosome contained a photoreceptor nucleus (labeled with DAPI, arrow) that was identified as a rod photoreceptor by rhodopsin immunopositivity (superposition of Iba1+ phagosome with rhodopsin+ soma in orthogonal views). (B) Example of an amoeboid microglia in the ONL with multiple phagosomes containing both rhodopsin-positive (arrowhead) and rhodopsin-negative (arrow) nuclei. (C) Rhodopsin+ nuclei can be localized within CD68-positive phagosomes in infiltrating microglia, indicating phagocytic engulfment of rods. Scale bar, 10 μm.

D, E The peak of microglial phagocytic activity occurred around P21 and declined subsequently, as measured by the number of rods phagocytosed (D) and the number of photoreceptors phagocytosed per microglial cell (E) (n = 3 animals at each time point).

F–J Infiltrating microglia phagocytose TUNEL-negative rods in the ONL. (F) Although the peaks of TUNEL positivity and microglial phagocytosis occurred concurrently, the majority of rod photoreceptors undergoing microglial phagocytosis were non-apoptotic (arrows), while many apoptotic photoreceptors were not phagocytosed by microglia (arrowheads); representative example of a P21 retina demonstrates non-overlapping patterns of TUNEL positivity (red) and microglial phagocytosis (Iba1, green). (G) Confocal analysis of a representative amoeboid microglial cell demonstrates that while the photoreceptor nuclei in phagosomes were rhodopsin-positive (white), they were mostly TUNEL-negative. Only a small fraction of microglial phagosomes contained TUNEL+, rhodopsin+ rods (example shown in H). Scale bars, 20 μm. (I) Quantitative analysis of photoreceptor nuclei within microglial phagosomes according to rhodopsin (black symbols), and TUNEL labeling (red symbols) at different times during rod degeneration. (J) Quantitative analysis of all photoreceptors undergoing either apoptosis or phagocytosis at P21–23 demonstrates that TUNEL+ apoptotic photoreceptors and phagocytosed photoreceptors consist of two distinct populations, indicating separate but parallel mechanisms of rod degeneration. Quantitative analyses involved retinal sections from n = 3 animals at each age.

Data information: Data points in (D, E, I) and column heights in (J) indicate mean, error bars indicate ± SEM.
Microglial phagocytosis of living photoreceptors contributes to inherited retinal degeneration. (2015)
Figure 4

Microglial phagocytosis of photoreceptors in alternative mouse models of retinitis pigmentosa (RP) and in human histopathological specimens

A–C Evidence of microglial phagocytosis of photoreceptors in other mouse models of RP. Histological analysis of a rd1 mouse retina (A) demonstrates microglial phagocytosis of photoreceptor nuclei in vibratome sections (left) and in flat-mounted retina (right). Phagocytosed nuclei were predominantly negative for TUNEL staining (arrows). Similar findings were found in the retinas of the rd16 mouse (loss-of-function mutation in the photoreceptor-expressed CEP290 gene, 1 month old) (B) and the RPGRIP-deficient mouse (6 months old) (C). Scale bar, 20 μm.

D–G Evidence of microglial phagocytosis of photoreceptors in other human histopathological specimens of RP. (D) Retinal section from a 30-year-old male donor with autosomal recessive RP (AR RP) showing extensive microglial infiltration of the ONL; expanded inset (right) shows multiple photoreceptor nuclei in phagosomes that were predominantly negative for TUNEL staining. (E, F) Retinal sections from two separate donors with autosomal dominant RP (AD RP1 = 68-year-old man, T17M rhodopsin mutation, AD RP1 = 50-year-old woman, Q-64-ter rhodopsin mutation) showing similar evidence of microglial phagocytosis. (G) Retinal section from a 46-year-old male donor with X-linked RP. Arrowheads indicate phagocytosed photoreceptor nuclei. Scale bars, 20 μm.