TIFA - KO cells were transfected with His ‑ tagged recombinant TIFA protein 24 h prior to H . pylori infection . Co ‑ IP with an anti ‑ His antibody was performed . Eluates and total cell lysates were analyzed by immunoblotting using the indicated antibodies . Asterisk denotes an unspecific band and the arrow indicates the TRAF2 band .

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TIFA-KO cells were transfected with His‑tagged recombinant TIFA protein 24 h prior to H. pylori infection. Co‑IP with an anti‑His antibody was performed. Eluates and total cell lysates were analyzed by immunoblotting using the indicated antibodies. Asterisk denotes an unspecific band and the arrow indicates the TRAF2 band.


AGS and ALPK1 - KO cells were infected with H . pylori for the times shown . Co ‑ IP with an anti ‑ TRAF6 antibody or isotype ‑ matched antibody ( IgG ) was performed . Eluates and total cell lysates were analyzed by immunoblotting using the indicated antibodies .

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AGS and ALPK1-KO cells were infected with H. pylori for the times shown. Co‑IP with an anti‑TRAF6 antibody or isotype‑matched antibody (IgG) was performed. Eluates and total cell lysates were analyzed by immunoblotting using the indicated antibodies.


AGS and TIFA - KO cells were infected with H . pylori for the times shown . Co ‑ IP with an anti ‑ TRAF6 antibody or isotype ‑ matched antibody ( IgG ) was performed . Eluates and total cell lysates were analyzed by immunoblotting using the indicated antibodies .

AGS and TIFA-KO cells were infected with H. pylori for the times shown. Co‑IP with an anti‑TRAF6 antibody or isotype‑matched antibody (IgG) was performed. Eluates and total cell lysates were analyzed by immunoblotting using the indicated antibodies.


AGS and TIFA - KO cells were treated with 30 ng / ml IL - 1β for the times shown . Co ‑ IP with an anti ‑ TRAF6 antibody or isotype ‑ matched antibody ( IgG ) was performed . Eluates and total cell lysates were analyzed by immunoblotting using the indicated antibodies .

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AGS and TIFA-KO cells were treated with 30 ng/ml IL-1β for the times shown. Co‑IP with an anti‑TRAF6 antibody or isotype‑matched antibody (IgG) was performed. Eluates and total cell lysates were analyzed by immunoblotting using the indicated antibodies.


TIFA - KO cells were transfected with His ‑ tagged recombinant TIFA protein 24 h prior to H . pylori infection or treatment with 30 ng / ml LTα1β2 . Co ‑ IP with an anti ‑ His antibody was performed . Eluates and total cell lysates were analyzed by immunoblotting using the indicated antibodies .

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TIFA-KO cells were transfected with His‑tagged recombinant TIFA protein 24 h prior to H. pylori infection or treatment with 30 ng/ml LTα1β2. Co‑IP with an anti‑His antibody was performed. Eluates and total cell lysates were analyzed by immunoblotting using the indicated antibodies.


AGS cells were pre ‑ incubated with 100 μM dynamin GTPase inhibitor Dynasore for 60 min followed by addition of ADP - heptose ( 200 nM ) . Co ‑ IP with an anti ‑ TIFA antibody was performed . Eluates and total cell lysates were analyzed by immunoblotting using the indicated antibodies . Asterisk indicates an unspecific band and the arrow indicates the TRAF3 band .

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AGS cells were pre‑incubated with 100 μM dynamin GTPase inhibitor Dynasore for 60 min followed by addition of ADP-heptose (200 nM). Co‑IP with an anti‑TIFA antibody was performed. Eluates and total cell lysates were analyzed by immunoblotting using the indicated antibodies. Asterisk indicates an unspecific band and the arrow indicates the TRAF3 band.


AGS and ALPK1 - KO cells were infected with H . pylori for the times shown . Co ‑ IP with an anti ‑ TIFA antibody or isotype ‑ matched antibody ( IgG ) was performed . Eluates and total cell lysates were analyzed by immunoblotting using the indicated antibodies .

AGS and ALPK1-KO cells were infected with H. pylori for the times shown. Co‑IP with an anti‑TIFA antibody or isotype‑matched antibody (IgG) was performed. Eluates and total cell lysates were analyzed by immunoblotting using the indicated antibodies.


AGS and TIFA - KO cells were infected with H . pylori for the times shown . Co ‑ IP with an anti ‑ TRAF2 antibody or isotype ‑ matched antibody ( IgG ) was performed . Eluates and total cell lysates were analyzed by immunoblotting using the indicated antibodies .

AGS and TIFA-KO cells were infected with H. pylori for the times shown. Co‑IP with an anti‑TRAF2 antibody or isotype‑matched antibody (IgG) was performed. Eluates and total cell lysates were analyzed by immunoblotting using the indicated antibodies.


AGS cells were transfected with siRNA against cIAP1 . Co ‑ IP with an anti ‑ TIFA antibody or isotype ‑ matched antibody ( IgG ) was performed . Eluates and total cell lysates were analyzed by immunoblotting using the indicated antibodies .

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AGS cells were transfected with siRNA against cIAP1. Co‑IP with an anti‑TIFA antibody or isotype‑matched antibody (IgG) was performed. Eluates and total cell lysates were analyzed by immunoblotting using the indicated antibodies.


TIFA - KO cells were transfected with empty vector plasmid ( pCMV6 ) , wild ‑ type human ( phTIFA ) , phosphorylation ‑ defective mutant ( pT9A ) or TRAF6 binding ‑ defective mutant ( pE178A ) FLAG ‑ tagged TIFA plasmids . Total cell lysates were analyzed by immunoblotting using the indicated antibodies .

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TIFA-KO cells were transfected with empty vector plasmid (pCMV6), wild‑type human (phTIFA), phosphorylation‑defective mutant (pT9A) or TRAF6 binding‑defective mutant (pE178A) FLAG‑tagged TIFA plasmids. Total cell lysates were analyzed by immunoblotting using the indicated antibodies.


TIFA - KO cells were transfected with empty vector plasmid ( pCMV6 ) , wild ‑ type human ( phTIFA ) , phosphorylation ‑ defective mutant ( pT9A ) or TRAF6 binding ‑ defective mutant ( pE178A ) FLAG ‑ tagged TIFA plasmids . Total cell lysates were separated by native gel electrophoresis under non - reducing conditions .

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TIFA-KO cells were transfected with empty vector plasmid (pCMV6), wild‑type human (phTIFA), phosphorylation‑defective mutant (pT9A) or TRAF6 binding‑defective mutant (pE178A) FLAG‑tagged TIFA plasmids. Total cell lysates were separated by native gel electrophoresis under non-reducing conditions.


In TIFA ‑ KO cells transiently expressing TIFA ‑ tdTomato , co - localization of TRAF6 ( A ) , TRAF2 ( B ) , cIAP1 ( C ) and TRAF3 ( D ) with TIFA - tdTomato , which formed TIFAsomes upon infection with H . pylori P1 wild ‑ type strain , were detected by immunofluorescence staining . The nuclei were counterstained with DAPI . Scale bar = 10 µm . TIFA ‑ KO cells transiently expressing TIFA ‑ tdTomato were infected with H . pylori P1 strain mutated in the gmhA gene ( ∆ HP0857 ) . Scale bar = 10 µm .

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In TIFA‑KO cells transiently expressing TIFA‑tdTomato, co-localization of TRAF6 (A), TRAF2 (B), cIAP1 (C) and TRAF3 (D) with TIFA-tdTomato, which formed TIFAsomes upon infection with H. pylori P1 wild‑type strain, were detected by immunofluorescence staining. The nuclei were counterstained with DAPI. Scale bar = 10 µm. TIFA‑KO cells transiently expressing TIFA‑tdTomato were infected with H. pylori P1 strain mutated in the gmhA gene (∆HP0857). Scale bar = 10 µm.


( c - f ) The indicated marker genes were used to select clusters for subsetting into stromal cells ( c ) , epithelial cells ( d ) , endothelial cells ( e ) , and leukocytes ( f ) .

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(c-f) The indicated marker genes were used to select clusters for subsetting into stromal cells (c), epithelial cells (d), endothelial cells (e), and leukocytes (f).


( g - j ) The heatmaps show the relative gene expression levels for the indicated marker genes for the indicated stromal ( g ) , epithelial ( h ) , endothelial ( i ) , and leukocyte ( j ) cell types .

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(g-j) The heatmaps show the relative gene expression levels for the indicated marker genes for the indicated stromal (g), epithelial (h), endothelial (i), and leukocyte (j) cell types.


( d ) Differential gene expression between end stage lung disease patients and controls across cohorts was compared for the indicated cell type identities . The color code demonstrates similarities of gene expression changes calculated by Pearson correlation of the t - value coefficient , which represents differences in the likelihood of detection for any gene between health and disease .

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(d) Differential gene expression between end stage lung disease patients and controls across cohorts was compared for the indicated cell type identities. The color code demonstrates similarities of gene expression changes calculated by Pearson correlation of the t-value coefficient, which represents differences in the likelihood of detection for any gene between health and disease.


( e ) The heatmap shows the top 79 genes differentially expressed in the indicated cell type identities .

(e) The heatmap shows the top 79 genes differentially expressed in the indicated cell type identities.


( f - h ) The box plots illustrate differences in mRNA detection for the indicated genes between tissues from end stage lung fibrosis patients in ( f ) alveolar epithelial cells , ( g ) fibroblasts , and ( h ) macrophages when compared to control tissue . The boxes represent the interquartile range , the horizontal line in the box is the median , and the whiskers represent 1 . 5 times the interquartile range ( Chicago cohort : ILD n = 9 , controls n = 8 ; Nashville cohort : ILD n = 20 , controls n = 10 ; Munich cohort : ILD n = 3 , controls n = 11 ) .

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(f-h) The box plots illustrate differences in mRNA detection for the indicated genes between tissues from end stage lung fibrosis patients in (f) alveolar epithelial cells, (g) fibroblasts, and (h) macrophages when compared to control tissue. The boxes represent the interquartile range, the horizontal line in the box is the median, and the whiskers represent 1.5 times the interquartile range (Chicago cohort: ILD n = 9, controls n = 8; Nashville cohort: ILD n = 20, controls n = 10; Munich cohort: ILD n = 3, controls n = 11).


( e ) The heatmap shows changes of the indicated cell type signatures in published bulk RNA - seq data ( GEO GSE124685 ) across different histopathological stages that represent increasing extent of fibrosis from stage 1 - 3 , as determined by quantitative micro - CT imaging and tissue histology Samples used in this study were 10 IPF and 6 control patients .

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(e) The heatmap shows changes of the indicated cell type signatures in published bulk RNA-seq data (GEO GSE124685) across different histopathological stages that represent increasing extent of fibrosis from stage 1-3, as determined by quantitative micro-CT imaging and tissue histology Samples used in this study were 10 IPF and 6 control patients.


( f ) The heatmaps show z - scores for the individual marker genes of the indicated cell types across IPF stages and controls .

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(f) The heatmaps show z-scores for the individual marker genes of the indicated cell types across IPF stages and controls.


( b ) The boxplots show the number of proteins quantified ( y - axis ) across various diagnoses ( x - axis ) . The mean and 10 - 90 percentiles are shown ; the dotted line marks 1000 proteins ( IPF n = 16 , HP / EAA n = 8 , COP n = 11 , NSIP n = 10 , RB - IL , n = 3 , Sarcoidosis n = 22 , other ILDs n = 25 , non - IL , n = 29 ) .

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(b) The boxplots show the number of proteins quantified (y-axis) across various diagnoses (x-axis). The mean and 10-90 percentiles are shown; the dotted line marks 1000 proteins (IPF n=16, HP/EAA n=8, COP n=11, NSIP n=10, RB-IL, n=3, Sarcoidosis n=22, other ILDs n=25, non-IL, n=29).


( c Empirical cumulative density plots depict the distribution of correlation coefficients for ( c ) Myofibroblast markers ( red points ) with DLCO in comparison to all background proteins ( black line ) .

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(c Empirical cumulative density plots depict the distribution of correlation coefficients for (c) Myofibroblast markers (red points) with DLCO in comparison to all background proteins (black line).


d ) Empirical cumulative density plots depict the distribution of correlation coefficients for ( d ) Plasma cell markers ( red points ) with % alveolar macrophages in BAL in comparison to all background proteins ( black line ) .

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d) Empirical cumulative density plots depict the distribution of correlation coefficients for (d) Plasma cell markers (red points) with % alveolar macrophages in BAL in comparison to all background proteins (black line).


( e ) Barplot displays coefficients derived from correlating lung function associations at the protein level with cell type specific ILD associations at the RNA level .

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(e) Barplot displays coefficients derived from correlating lung function associations at the protein level with cell type specific ILD associations at the RNA level.


( f ) The heatmap illustrates gene expression changes associated with lung fibrosis across indicated cell types ( columns ) for selected BALF protein biomarkers ( rows ) . The dotplot visualizes the frequency changes of the indicated cell types inferred from the deconvolution of bulk mRNA data of ILD samples compared to controls ( GSE47460 ) . Samples used in this study from the LTRC n = 254 ILD patients and n = 108 controls .

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(f) The heatmap illustrates gene expression changes associated with lung fibrosis across indicated cell types (columns) for selected BALF protein biomarkers (rows). The dotplot visualizes the frequency changes of the indicated cell types inferred from the deconvolution of bulk mRNA data of ILD samples compared to controls (GSE47460). Samples used in this study from the LTRC n = 254 ILD patients and n = 108 controls.


( a , b ) Scatter plots stratify genes based on the protein lung function associations ( y - axis ) and cell type specific ILD associations ( x - axis ) . The size of the dots represents the detection level of each gene in the corresponding cell type . Colors highlight genes with marginal associations at the protein and RNA levels .

(a, b) Scatter plots stratify genes based on the protein lung function associations (y-axis) and cell type specific ILD associations (x-axis). The size of the dots represents the detection level of each gene in the corresponding cell type. Colors highlight genes with marginal associations at the protein and RNA levels.


( c , d ) The box plots illustrate differences in mRNA detection for the indicated genes between tissues from end stage lung fibrosis patients in ( c ) basal cells , and ( d ) alveolar epithelial cells when compared to controls . The boxes represent the interquartile range , the horizontal line in the box is the median , and the whiskers represent 1 . 5 times the interquartile range ( Chicago cohort : ILD n = 9 , controls n = 8 ; Nashville cohort : ILD n = 20 , controls n = 10 ; Munich cohort : ILD n = 3 , controls n = 11 ) .

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(c, d) The box plots illustrate differences in mRNA detection for the indicated genes between tissues from end stage lung fibrosis patients in (c) basal cells, and (d) alveolar epithelial cells when compared to controls. The boxes represent the interquartile range, the horizontal line in the box is the median, and the whiskers represent 1.5 times the interquartile range (Chicago cohort: ILD n = 9, controls n = 8; Nashville cohort: ILD n = 20, controls n = 10; Munich cohort: ILD n = 3, controls n = 11).


( e , f ) The scatter plots show the positive correlation of the indicated proteins in BALF ( MS - intensity , x - axis ) with meta lung function ( y - axis ) .

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(e, f) The scatter plots show the positive correlation of the indicated proteins in BALF (MS-intensity, x-axis) with meta lung function (y-axis).


( g - j ) Immunofluorescence analysis of the indicated proteins and cell type markers in IPF ( n = 3 ) and control samples ( n = 3 ) .

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(g-j) Immunofluorescence analysis of the indicated proteins and cell type markers in IPF (n=3) and control samples (n=3).


( a ) The scatter plots show the positive correlation of CFHR1 in BALF ( MS - intensity , x - axis ) with meta lung function ( y - axis ) .

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(a) The scatter plots show the positive correlation of CFHR1 in BALF (MS-intensity, x-axis) with meta lung function (y-axis).


( c ) The volcano plot depicts the fold changes ( x - axis ) and the - log10 p - values ( y - axis ) derived from the differential gene expression analysis using diffxpy between ILD and controls within the subset of PDGFRB + pericytes ( outlier values truncated ) .

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(c) The volcano plot depicts the fold changes (x-axis) and the -log10 p-values (y-axis) derived from the differential gene expression analysis using diffxpy between ILD and controls within the subset of PDGFRB+ pericytes (outlier values truncated).


( e ) The heatmap shows the expression of genes most strongly associated with SSTR2 expression within the pericytes ( left color bar indicates significant correlation or anti - correlation ) .

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(e) The heatmap shows the expression of genes most strongly associated with SSTR2 expression within the pericytes (left color bar indicates significant correlation or anti-correlation).


( f ) Immunofluorescence analysis of SSTR2 , the pericyte cell type marker PDGFRB and a marker for smooth muscle cells DESMIN in IPF ( n = 3 ) and control samples ( n = 3 ) .

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(f) Immunofluorescence analysis of SSTR2, the pericyte cell type marker PDGFRB and a marker for smooth muscle cells DESMIN in IPF (n=3) and control samples (n=3).


( g ) Representative images of immunohistochemistry analysis of SSTR2 protein expression in tissue regions ( n = 474 ) from ILD patients ( n = 53 ) and control patients ( n = 26 ) .

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(g) Representative images of immunohistochemistry analysis of SSTR2 protein expression in tissue regions (n = 474) from ILD patients (n = 53) and control patients (n = 26).


( h ) Correlation of immunohistochemistry signal of SSTR2 with Ashcroft scores .

(h) Correlation of immunohistochemistry signal of SSTR2 with Ashcroft scores.


( i ) The bar graph shows the genes most strongly correlated with SSTR2 belonging to the GO category " transcription regulators " . The dotted line marks a correlation coefficient of zero .

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(i) The bar graph shows the genes most strongly correlated with SSTR2 belonging to the GO category "transcription regulators". The dotted line marks a correlation coefficient of zero.


( c ) Relative expression level of CRTAC1 across human organs .

(c) Relative expression level of CRTAC1 across human organs.


( d ) The box plots illustrate differences in mRNA detection for CRTAC1 in alveolar epithelial cells from fibrosis patients compared to control samples across the three indicated patient cohorts ( Chicago cohort : ILD n = 9 , controls n = 8 ; Nashville cohort : ILD n = 20 , controls n = 10 ; Munich cohort : ILD n = 3 , controls n = 11 ) .

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(d) The box plots illustrate differences in mRNA detection for CRTAC1 in alveolar epithelial cells from fibrosis patients compared to control samples across the three indicated patient cohorts (Chicago cohort: ILD n = 9, controls n = 8; Nashville cohort: ILD n = 20, controls n = 10; Munich cohort: ILD n = 3, controls n = 11).


( e ) Relative gene expression levels of CRTAC1 in GSE47460 . Dots represent average expression in the tissue of individual patients . The line represents the mean , error bars show SD . CRTAC1 is significantly downregulated in ILD but not COPD

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(e) Relative gene expression levels of CRTAC1 in GSE47460. Dots represent average expression in the tissue of individual patients. The line represents the mean, error bars show SD. CRTAC1 is significantly downregulated in ILD but not COPD patients


( f ) For each single cell cohort , the gene - gene correlations with CRTAC1 within the SFTPC + AT - 2 cells were calculated . The indicated genes were selected based on their common direction of correlation across cohorts .

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(f) For each single cell cohort, the gene-gene correlations with CRTAC1 within the SFTPC+ AT-2 cells were calculated. The indicated genes were selected based on their common direction of correlation across cohorts.


( h ) The bar graph shows the gene categories most strongly correlated with CRTAC1 belonging to the GO category of " transcription regulators " . The dotted line marks a correlation coefficient of zero .

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(h) The bar graph shows the gene categories most strongly correlated with CRTAC1 belonging to the GO category of "transcription regulators". The dotted line marks a correlation coefficient of zero.


( n ) Immunofluorescence analysis of SPRR1A , KRT8 as well as SFTPC in IPF ( n = 3 ) and control samples ( n = 2 ) .

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(n) Immunofluorescence analysis of SPRR1A, KRT8 as well as SFTPC in IPF (n=3) and control samples (n=2).


( o ) A high throughput experimental workflow for plasma proteomics ( Niu allowed for profiling of two independent cohorts of ILD patients ( Munich , n = 30 and Hannover , n = 81 ; healthy age matched controls , n = 30 ) . All proteins quantified in plasma are shown , ranked by their abundance measured by mass spectrometry ( MS - intensity ) .

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(o) A high throughput experimental workflow for plasma proteomics(Niu allowed for profiling of two independent cohorts of ILD patients (Munich, n=30 and Hannover, n=81; healthy age matched controls, n=30). All proteins quantified in plasma are shown, ranked by their abundance measured by mass spectrometry (MS-intensity).


( p ) The indicated proteins from the plasma analysis were selected based on their common direction of correlation with patient lung function in two independent patient cohorts with distinct clinical characteristics .

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(p) The indicated proteins from the plasma analysis were selected based on their common direction of correlation with patient lung function in two independent patient cohorts with distinct clinical characteristics.


( q ) The heatmap shows the predicted relative contribution of lung cell types to the association of protein biomarker signatures in plasma with lung function ( forced vital capacity - FVC ) . Patients were split in two groups , one with a mild decline in lung function [ FVC 60 - 100 % ] and one with severe loss of lung function [ FVC 20 - 60 % ] and compared to healthy age matched controls .

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(q) The heatmap shows the predicted relative contribution of lung cell types to the association of protein biomarker signatures in plasma with lung function (forced vital capacity - FVC). Patients were split in two groups, one with a mild decline in lung function [FVC 60-100%] and one with severe loss of lung function [FVC 20-60%] and compared to healthy age matched controls.


( B ) IHC for CDC50A and RIM1 on cultured neurons at DIV21 . The left panel shows CDC50A is expressed in both neuronal soma and processes . The right panel exhibits CDC50A colocalize to pre - synapses alongside neuronal processes . MAP2 , Microtubule - associated protein 2 , was used to identify neuronal cells and trace dendritic processes . RIM1 is an active zone protein , which marks pre - synapses . Left scale bar = 50 µm ; right scale bar = 2 µm . ( C ) Quantification of the percentage of RIM1 + pre - synapses colocalizing to CDC50A . N = 3 for biological replicates .

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(B) IHC for CDC50A and RIM1 on cultured neurons at DIV21. The left panel shows CDC50A is expressed in both neuronal soma and processes. The right panel exhibits CDC50A colocalize to pre-synapses alongside neuronal processes. MAP2, Microtubule-associated protein 2, was used to identify neuronal cells and trace dendritic processes. RIM1 is an active zone protein, which marks pre-synapses. Left scale bar = 50 µm; right scale bar = 2 µm. (C) Quantification of the percentage of RIM1+ pre-synapses colocalizing to CDC50A. N = 3 for biological replicates.


( E ) IHC for CDC50A and vGluT2 in the developmental dLGN at P14 , P21 and P30 . The left panels show the overview of immunostaining in the dLGN , scale bar = 200 µm . The right panels are the zoomed - in images , scale bar = 5 µm . Arrows indicate the colocalized CDC50A and vGluT2 . ( F ) Quantification of the percentage of CDC50A + / vGlut2 + synapses . The result shows that a significantly higher percentage of CDC50A + / vGlut2 + synapses at P21 and P30 than P14 . N = 6 for biological replicates .

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(E) IHC for CDC50A and vGluT2 in the developmental dLGN at P14, P21 and P30. The left panels show the overview of immunostaining in the dLGN, scale bar = 200 µm. The right panels are the zoomed-in images, scale bar = 5 µm. Arrows indicate the colocalized CDC50A and vGluT2. (F) Quantification of the percentage of CDC50A+/vGlut2+ synapses. The result shows that a significantly higher percentage of CDC50A+/vGlut2+ synapses at P21 and P30 than P14. N = 6 for biological replicates.


( C ) IHC for CDC50A . CDC50A protein intensities were reduced after neuronal inactivation ( day 7 ) , and was recovered after removing neuronal inhibitors ( day 13 ) . Scale bar , 10 µm . ( D ) Relative CDC50A intensities of neurons . N = 5 for biological replicates , mean ± SD , * p < 0 . 5 , * * * * p < 0 . 0001 by one - way ANOVA with Holm - Sidak ' s multiple comparisons test .

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(C) IHC for CDC50A. CDC50A protein intensities were reduced after neuronal inactivation (day 7), and was recovered after removing neuronal inhibitors (day 13). Scale bar, 10 µm. (D) Relative CDC50A intensities of neurons. N = 5 for biological replicates, mean ± SD, * p < 0.5, **** p < 0.0001 by one-way ANOVA with Holm-Sidak's multiple comparisons test.


( E ) CDC50A expression at RGC inputs after 5 - day consecutive TTX treatment . Scale bar , 10 µm . ( F ) Quantification of the percentage of CDC50A + / vGlut2 + synapses . N = 5 for biological replicates , mean ± SD , * * * * p < 0 . 0001 by Student ' s test .

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(E) CDC50A expression at RGC inputs after 5-day consecutive TTX treatment. Scale bar, 10 µm. (F) Quantification of the percentage of CDC50A+/vGlut2+ synapses. N = 5 for biological replicates, mean ± SD, **** p < 0.0001 by Student's test.


( A ) Immunostaining of CDC50A on Cdc50a knockdown neurons and controls . Accell Cdc50a siRNA pool was used to knock down Cdc50a in neurons for 7 days . CDC50A expression alongside neuronal processed was reduced after Cdc50a siRNA treatment . Scale bar , 10 µm .

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(A) Immunostaining of CDC50A on Cdc50a knockdown neurons and controls. Accell Cdc50a siRNA pool was used to knock down Cdc50a in neurons for 7 days. CDC50A expression alongside neuronal processed was reduced after Cdc50a siRNA treatment. Scale bar, 10 µm.


( D ) IHC of Cdc50a - RNAi neurons exhibited pSIVA colocalized with synapsin I , a general synapse marker . The dotted square was zoomed - in shown on the right side . Arrows indicated where pSIVA colocalized with synapsin I . Scale bar , 5 µm .

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(D) IHC of Cdc50a-RNAi neurons exhibited pSIVA colocalized with synapsin I, a general synapse marker. The dotted square was zoomed-in shown on the right side. Arrows indicated where pSIVA colocalized with synapsin I. Scale bar, 5 µm.


( D ) Representative IHC images of CDC50A expression at synapses . Scale bar , 5 µm . Arrows indicate VGluT2 + RGC inputs . ( E ) Relative CDC50A expression at mCherry + synapses in Cdc50a RNAi mice and controls . N = 3 for biological replicates , mean ± SD , * p < 0 . 05 by Student ' s t - test .

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(D) Representative IHC images of CDC50A expression at synapses. Scale bar, 5 µm. Arrows indicate VGluT2+ RGC inputs. (E) Relative CDC50A expression at mCherry+ synapses in Cdc50a RNAi mice and controls. N = 3 for biological replicates, mean ± SD, * p < 0.05 by Student's t-test.


( F ) PSIVA staining in the dLGN . Exposed PS was detected by pSIVA on RGC inputs in Cdc50a - RNAi mice . Scale bar , 5 µm . Arrows indicate pSIVA + / mCherry + RGC inputs . ( G ) Quantification of the percentage of pSIVA + / mCherry + RGC inputs in total mCherry + RGC inputs . N = 4 for biological replicates , mean ± SEM , * * p < 0 . 01 by Student ' s t - test .

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(F) PSIVA staining in the dLGN. Exposed PS was detected by pSIVA on RGC inputs in Cdc50a-RNAi mice. Scale bar, 5 µm. Arrows indicate pSIVA+/mCherry+ RGC inputs. (G) Quantification of the percentage of pSIVA+/mCherry+ RGC inputs in total mCherry+ RGC inputs. N = 4 for biological replicates, mean ± SEM, **p < 0.01 by Student's t-test.


( F ) Maximum intensity z - projection images of vGluT2 + synapses in the core region of Cdc50a RNAi mice and controls . Right panel showed enlarged synapses in the boxed region . Scale bar , 20 µm .

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(F) Maximum intensity z-projection images of vGluT2+ synapses in the core region of Cdc50a RNAi mice and controls. Right panel showed enlarged synapses in the boxed region. Scale bar, 20 µm.


( H ) Maximum intensity z - projection images of vGluT2 + synapses in the shell region . Enlarged images ( right panel ) shows large " complex encapsulated " boutons . Scale bar , 20 µm . ( I ) Quantification of vGluT2 + synapse density . N = 3 for biological replicates , mean ± SD , Student ' s t - test .

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(H) Maximum intensity z-projection images of vGluT2+ synapses in the shell region. Enlarged images (right panel) shows large "complex encapsulated" boutons. Scale bar, 20 µm. (I) Quantification of vGluT2+ synapse density. N = 3 for biological replicates, mean ± SD, Student's t-test.


( D ) Images of CTB488 and CTB647 labeling of RGC inputs in the dLGN . Scale bar , 20 µm . ( E ) Quantification of CTB488 + inputs ( non - infected areas ) versus CTB647 + inputs ( infected areas ) in Cdc50a RNAi mice and controls . N = 3 for biological replicates , mean ± SEM , * * * * p < 0 . 0001 , two - way ANOVA with Sidak ' s multiple comparisons test .

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(D) Images of CTB488 and CTB647 labeling of RGC inputs in the dLGN. Scale bar, 20 µm. (E) Quantification of CTB488+ inputs (non-infected areas) versus CTB647+ inputs (infected areas) in Cdc50a RNAi mice and controls. N = 3 for biological replicates, mean ± SEM, **** p < 0.0001, two-way ANOVA with Sidak's multiple comparisons test.


( A ) The confocal images , 3D - rendered images and orthogonal views of microglia engulfing vGluT2 + synapses in controls and Cdc50a - RNAi mice . Scale bar , 10 µm .

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(A) The confocal images, 3D-rendered images and orthogonal views of microglia engulfing vGluT2+ synapses in controls and Cdc50a-RNAi mice. Scale bar, 10 µm.


( A ) IHC images for vGluT2 staining in the dLGN of microglial Gpr56 conditional knockout mice ( Gpr56fl / fl ; Cx3cr1cre / + ) and controls ( Gpr56 + / + ; Cx3cr1cre / + ) . Both mice were intravitreally injected with AAV2 carrying Cdc50a shRNA . The yellow squares indicated the AAV2 non - infected areas and the dotted outlines indicated the AAV2 infected areas . Scale bar , 200 µm .

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(A) IHC images for vGluT2 staining in the dLGN of microglial Gpr56 conditional knockout mice (Gpr56fl/fl; Cx3cr1cre/+) and controls (Gpr56+/+; Cx3cr1cre/+). Both mice were intravitreally injected with AAV2 carrying Cdc50a shRNA. The yellow squares indicated the AAV2 non-infected areas and the dotted outlines indicated the AAV2 infected areas. Scale bar, 200 µm.


( B ) Confocal images of vGluT2 + synapses in the AAV2 non - infected areas and infected areas of the dLGN . Scale bar , 20 µm . ( C ) Quantification of vGluT2 + synapses in the non - infected areas and infected areas of microglial GPR56 conditional knockout mice and controls . N = 4 for biological replicates , mean ± SD , * p < 0 . 05 , * * * * p < 0 . 0001 by two - way ANOVA with Sidak ' s multiple comparisons test .

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(B) Confocal images of vGluT2+ synapses in the AAV2 non-infected areas and infected areas of the dLGN. Scale bar, 20 µm. (C) Quantification of vGluT2+ synapses in the non-infected areas and infected areas of microglial GPR56 conditional knockout mice and controls. N = 4 for biological replicates, mean ± SD, * p<0.05, **** p < 0.0001 by two-way ANOVA with Sidak's multiple comparisons test.


Verification of PTPδ - tdTomato reporter mice by Western blot analysis of whole - brain lysates from heterozygote ( HT ) and homozygous ( Ho ) mice ( P21 ) and PTPδ using mCherry antibodies . Note that levels of endogenous PTPδ protein ( ~ 85 kDa ) are decreased , and that PTPδ - tdTomato protein ( ~ 140 kDa ) is detectable only in reporter mice ( HT and Ho ) . Note also that the 85 - kDa band represents the transmembrane domain + cytoplasmic fragment of the full - length PTPδ protein that undergoes proteolytic cleavage at extracellular membrane - proximal sites , which can only be recognized by the PTPδ C - terminal antibody that targets the cytoplasmic region . The PTPδ - tdTomato protein ( ~ 140 kDa ) contains the membrane domain + cytoplasmic fragment of PTPδ ( ~ 85 kDa ) fused to tdTomato ( 55 kDa ) . The N - terminal antibody recognizes equal amounts of the C - terminal cleavage product derived from PTPδ and PTPδ - tdTomato proteins .

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Verification of PTPδ-tdTomato reporter mice by Western blot analysis of whole-brain lysates from heterozygote (HT) and homozygous (Ho) mice (P21) and PTPδ using mCherry antibodies. Note that levels of endogenous PTPδ protein (~85 kDa) are decreased, and that PTPδ-tdTomato protein (~140 kDa) is detectable only in reporter mice (HT and Ho). Note also that the 85-kDa band represents the transmembrane domain + cytoplasmic fragment of the full-length PTPδ protein that undergoes proteolytic cleavage at extracellular membrane-proximal sites, which can only be recognized by the PTPδ C-terminal antibody that targets the cytoplasmic region. The PTPδ-tdTomato protein (~140 kDa) contains the membrane domain + cytoplasmic fragment of PTPδ (~85 kDa) fused to tdTomato (55 kDa). The N-terminal antibody recognizes equal amounts of the C-terminal cleavage product derived from PTPδ and PTPδ-tdTomato proteins.


Representative coronal , horizontal , and sagittal sections from PTPδ - tdTomato mice . Enlarged windows are demarcated by black dotted lines ( Fig EV1A - C ) . Red tdTomato images were converted to grayscale images for clarity . ACA , anterior cingulate area ; ACB , nucleus accumbens ; BLA , basolateral amygdala ; BMA , basomedial amygdala ; CA1 , cornu ammonis area 1 ; CA2 , cornu ammonis area 2 ; CA3 , cornu ammonis area 3 ; CB , cerebellum ; CC , corpus callosum ; CEA , central amygdala ; CP , caudate putamen ; CST , corticospinal tract ; DG , dentate gyrus ; EP , endopiriform nucleus , HY , hypothalamus ; L1 , cortical layer 1 ; L2 / 3 , cortical layer 2 / 3 ; LA , lateral amygdala ; lENT , lateral entorhinal area ; LH , lateral habenula ; MB , midbrain ; MEA , medial amygdala ; mENT , medial entorhinal area ; MH , medial habenula ; PIR1 , piriform molecular layer ; PIR2 , piriform pyramidal layer ; PIR3 , piriform polymorph layer ; PL , prelimbic ; PONS , pons ; PRE , presubiculum ; RSP , retrosplenial area ; SS , somatosensory area ; SUB , subiculum ; TEa , temporal association area ; TH , thalamus ; VIS , visual area ; Scale bar , 500 μm .

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Representative coronal, horizontal, and sagittal sections from PTPδ-tdTomato mice. Enlarged windows are demarcated by black dotted lines (Fig EV1A-C). Red tdTomato images were converted to grayscale images for clarity. ACA, anterior cingulate area; ACB, nucleus accumbens; BLA, basolateral amygdala; BMA, basomedial amygdala; CA1, cornu ammonis area 1; CA2, cornu ammonis area 2; CA3, cornu ammonis area 3; CB, cerebellum; CC, corpus callosum; CEA, central amygdala; CP, caudate putamen; CST, corticospinal tract; DG, dentate gyrus; EP, endopiriform nucleus, HY, hypothalamus; L1, cortical layer 1; L2/3, cortical layer 2/3; LA, lateral amygdala; lENT, lateral entorhinal area; LH, lateral habenula; MB, midbrain; MEA, medial amygdala; mENT, medial entorhinal area; MH, medial habenula; PIR1, piriform molecular layer; PIR2, piriform pyramidal layer; PIR3, piriform polymorph layer; PL, prelimbic; PONS, pons; PRE, presubiculum; RSP, retrosplenial area; SS, somatosensory area; SUB, subiculum; TEa, temporal association area; TH, thalamus; VIS, visual area; Scale bar, 500 μm.


PTPδ - tdTomato protein is detected in tau - positive axonal compartments , but not in MAP2 - positive dendritic compartments in cultured neurons ( entorhinal cortex + hippocampus ; 1 : 2 mixture ; days in vitro or DIV 24 ) derived from Ptprd - / - mouse embryos . Note that the tdTomato signals envelop , but not overlap with , MAP2 - delineated dendrites , although this does not necessarily suggest that tdTomato signals not in dendrites . Scale bars , 10 μm in main images and 2 μm in enlarged images .

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PTPδ-tdTomato protein is detected in tau-positive axonal compartments, but not in MAP2-positive dendritic compartments in cultured neurons (entorhinal cortex + hippocampus; 1:2 mixture; days in vitro or DIV 24) derived from Ptprd-/- mouse embryos. Note that the tdTomato signals envelop, but not overlap with, MAP2-delineated dendrites, although this does not necessarily suggest that tdTomato signals not in dendrites. Scale bars, 10 μm in main images and 2 μm in enlarged images.


Presynaptic PTPδ - tdTomato clusters colocalize more strongly with PSD - 95 clusters ( excitatory postsynaptic marker ) relative to gephyrin ( inhibitory postsynaptic marker ) clusters in cultured neurons ( entorhinal cortex + hippocampus ; DIV24 ) derived from Ptprd - / - mouse embryos . See Fig EV1F for the quantification of the results . Scale bars , 10 μm in main images and 2 μm in enlarged images .

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Presynaptic PTPδ-tdTomato clusters colocalize more strongly with PSD-95 clusters (excitatory postsynaptic marker) relative to gephyrin (inhibitory postsynaptic marker) clusters in cultured neurons (entorhinal cortex + hippocampus; DIV24) derived from Ptprd-/- mouse embryos. See Fig EV1F for the quantification of the results. Scale bars, 10 μm in main images and 2 μm in enlarged images.


Ultrastructural localization of PTPδ - tdTomato protein ( red arrows ; DAB staining ) at axon terminals juxtaposed to electron - dense postsynaptic densities ( purple arrows ; PSDs ) and also at vGlut2 - positive excitatory synaptic axon terminals ( green arrows ; immunogold staining ) but not at GAD67 / 65 - positive inhibitory synaptic axon terminals ( blue arrows ; immunogold staining ) in the stratum lacunosum - moleculare ( SLM ) region of the CA1 hippocampal region ( P21 ) . Note that ~ 85 % of PTPδ - tdTomato signals are present in excitatory presynaptic terminals apposed to PSDs , ~ 10 % at presynaptic terminals not apposed to PSDs , and ~ 5 % in neuropils other than presynaptic terminals . Axon terminals are indicated by pink shades . Scale bar , 500 nm . ( n = 4 areas from 2 mice [ WT and KO ] ) .

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Ultrastructural localization of PTPδ-tdTomato protein (red arrows; DAB staining) at axon terminals juxtaposed to electron-dense postsynaptic densities (purple arrows; PSDs) and also at vGlut2-positive excitatory synaptic axon terminals (green arrows; immunogold staining) but not at GAD67/65-positive inhibitory synaptic axon terminals (blue arrows; immunogold staining) in the stratum lacunosum-moleculare (SLM) region of the CA1 hippocampal region (P21). Note that ~85% of PTPδ-tdTomato signals are present in excitatory presynaptic terminals apposed to PSDs, ~10% at presynaptic terminals not apposed to PSDs, and ~5% in neuropils other than presynaptic terminals. Axon terminals are indicated by pink shades. Scale bar, 500 nm. (n = 4 areas from 2 mice [WT and KO]).


Strong PTPδ - tdTomato ( red ) signals in the SLM layer of the CA1 region and the MO layer of the DG in the hippocampus ( 2 months ) , revealed by quantitative analysis of PTPδ - tdTomato fluorescence across hippocampal lamina . DAPI was used for nuclear staining . CC , corpus callosum ; SO , stratum oriens ; SP , stratum pyramidale ; SR , stratum radiatum ; SLM , stratum lacunosum moleculare ; MO , molecular layer ; SG , granule cell layer ; PO , polymorph layer . Scale bar 500 μm .

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Strong PTPδ-tdTomato (red) signals in the SLM layer of the CA1 region and the MO layer of the DG in the hippocampus (2 months), revealed by quantitative analysis of PTPδ-tdTomato fluorescence across hippocampal lamina. DAPI was used for nuclear staining. CC, corpus callosum; SO, stratum oriens; SP, stratum pyramidale; SR, stratum radiatum; SLM, stratum lacunosum moleculare; MO, molecular layer; SG, granule cell layer; PO, polymorph layer. Scale bar 500 μm.


Biochemical enrichment of PTPδ protein in the hippocampal SLM layer relative to the SR layer . Micro - dissected SLM and SR lysates ( 2 months ) were immunoblotted for PTPδ ( C - terminal antibodies ) and α - tubulin ( control ) . The asterisk indicates a non - specific band . Analysis of immunoblots reveal significant enrichment of PTPδ in the SLM compared to the SR . ( n = 4 mice for SR and SLM , mean ± SEM , * * * p < 0 . 001 , Student ' s t - test ; See Dataset EV2 for statistical details for these and all following results ) .

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Biochemical enrichment of PTPδ protein in the hippocampal SLM layer relative to the SR layer. Micro-dissected SLM and SR lysates (2 months) were immunoblotted for PTPδ (C-terminal antibodies) and α-tubulin (control). The asterisk indicates a non-specific band. Analysis of immunoblots reveal significant enrichment of PTPδ in the SLM compared to the SR. (n = 4 mice for SR and SLM, mean ± SEM, ***p < 0.001, Student's t-test; See Dataset EV2 for statistical details for these and all following results).


Gene dose - dependent reductions in PTPδ protein levels in whole brains of Ptprd + / - ( HT ) and Ptprd - / - ( KO ) mice relative to Ptprd + / + ( WT ) mice ( P21 ) , shown by immunoblot analyses . Note that the C - terminal antibody also detects a non - specific band of similar size indicated by an asterisk , possibly either PTPσ or LAR .

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Gene dose-dependent reductions in PTPδ protein levels in whole brains of Ptprd+/- (HT) and Ptprd-/- (KO) mice relative to Ptprd+/+ (WT) mice (P21), shown by immunoblot analyses. Note that the C-terminal antibody also detects a non-specific band of similar size indicated by an asterisk, possibly either PTPσ or LAR.


Suppressed basal excitatory synaptic transmission in the Ptprd - / - hippocampal SLM layer , but not SO or SR layer ( 2 months ; male ) , as shown by the I / O ratio ( n = 14 slices from 5 mice [ WT - SO ] , 14 , 5 [ KO - SO ] , 11 , 4 [ WT - SR ] , 12 , 5 [ KO - SR ] , 14 , 6 [ WT - SLM ] , 12 , 6 [ KO - SLM ] , mean ± SEM , * p < 0 . 05 ; * * p < 0 . 01 , two - way repeated - measures / RM ANOVA , Holm - Sidak post - hoc multiple comparisons test ) . ( F , H , and J ) Normal presynaptic release in the Ptprd - / - hippocampal SLM , SO , and SR layers ( P22 - 27 ; male ) , as shown by the PPF ratio ( n = 13 slices from 5 mice [ WT - SO ] , 13 , 5 [ KO - SO ] , 10 , 4 [ WT - SR ] , 12 , 5 [ KO - SR ] , 15 , 6 [ WT - SLM ] , 14 , 6 [ KO - SLM ] , mean ± SEM , ns , not significant , two - way RM ANOVA ) .

O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Suppressed basal excitatory synaptic transmission in the Ptprd-/- hippocampal SLM layer, but not SO or SR layer (2 months; male), as shown by the I/O ratio (n = 14 slices from 5 mice [WT-SO], 14, 5 [KO-SO], 11, 4 [WT-SR], 12, 5 [KO-SR], 14, 6 [WT-SLM], 12, 6 [KO-SLM], mean ± SEM, *p < 0.05; **p < 0.01, two-way repeated-measures/RM ANOVA, Holm-Sidak post-hoc multiple comparisons test). (F, H, and J) Normal presynaptic release in the Ptprd-/- hippocampal SLM, SO, and SR layers (P22-27; male), as shown by the PPF ratio (n = 13 slices from 5 mice [WT-SO], 13, 5 [KO-SO], 10,4 [WT-SR], 12, 5 [KO-SR], 15, 6 [WT-SLM], 14, 6 [KO-SLM], mean ± SEM, ns, not significant, two-way RM ANOVA).


Decreased PSD density , but not PSD length , thickness , or perforation ( % ) , in the Ptprd - / - SLM ( P21 ; male ) , revealed by EM . Arrows in panel K indicate PSDs .

O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O

Decreased PSD density, but not PSD length, thickness, or perforation (%), in the Ptprd-/- SLM (P21; male), revealed by EM. Arrows in panel K indicate PSDs.


Decreased PSD density , but not PSD length , thickness , or perforation ( % ) , in the Ptprd - / - SLM ( P21 ; male ) , revealed by EM . Arrows in panel K indicate PSDs . ( n = 18 images from 3 mice [ WT and KO ] , * * * p < 0 . 001 , ns , not significant , Student ' s t - test for L , O - R , Mann - Whitney U test for M , N ) . Scale bar , 500 nm .

O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Decreased PSD density, but not PSD length, thickness, or perforation (%), in the Ptprd-/- SLM (P21; male), revealed by EM. Arrows in panel K indicate PSDs. (n = 18 images from 3 mice [WT and KO], ***p < 0.001, ns, not significant, Student's t-test for L, O-R, Mann-Whitney U test for M, N). Scale bar, 500 nm.


Decreased density of presynaptic nerve terminals but normal area of nerve terminals and normal density of presynaptic vesicles in the Ptprd - / - SLM ( P21 ; male ) , revealed by EM . ( n = 3 mice [ WT and KO ] , mean ± SEM , * * * p < 0 . 001 , ns , not significant , Student ' s t - test ) .

O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Decreased density of presynaptic nerve terminals but normal area of nerve terminals and normal density of presynaptic vesicles in the Ptprd-/- SLM (P21; male), revealed by EM. (n = 3 mice [WT and KO], mean ± SEM, ***p < 0.001, ns, not significant, Student's t-test).


A volcano plot of proteins that showed significant up - or downregulation of pTyr levels ( absolute fold change > 1 . 5 , p < 0 . 05 ) in the whole brain of Ptprd - / - mice ( P21 ) . Note that IL1RAPL1 is the protein with the strongest decrease in pTyr levels . ( n = 3 for WT and 3 for KO ) . See Dataset EV1 , ' Phosphoscan Results ' tab for a full list of significantly changed proteins .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A volcano plot of proteins that showed significant up- or downregulation of pTyr levels (absolute fold change > 1.5, p < 0.05) in the whole brain of Ptprd-/- mice (P21). Note that IL1RAPL1 is the protein with the strongest decrease in pTyr levels. (n = 3 for WT and 3 for KO). See Dataset EV1, 'Phosphoscan Results' tab for a full list of significantly changed proteins.


A volcano plot of presynaptic proteins included in the SynGO list to show that they are significantly changed in pTyr levels ( p < 0 . 05 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A volcano plot of presynaptic proteins included in the SynGO list to show that they are significantly changed in pTyr levels (p < 0.05).


A volcano plot of postsynaptic proteins included in the SynGO list to show that they are significantly changed in pTyr levels ( p < 0 . 05 ) .

A volcano plot of postsynaptic proteins included in the SynGO list to show that they are significantly changed in pTyr levels (p < 0.05).


Decreased pTyr levels , but normal total levels of IL1RAPL1 protein , in the Ptprd - / - brain ( P21 - 27 ) , as revealed by immunoprecipitation of IL1RAPL1 from whole - brain lysates followed by immunoblotting for total IL1RAPL1 protein and pTyr levels ( 4G10 antibody ) .

O O O O O O O O O B-MEASURED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O

Decreased pTyr levels, but normal total levels of IL1RAPL1 protein, in the Ptprd-/- brain (P21-27), as revealed by immunoprecipitation of IL1RAPL1 from whole-brain lysates followed by immunoblotting for total IL1RAPL1 protein and pTyr levels (4G10 antibody).


Decreased levels of synaptic IL1RAPL1 protein in the Ptprd - / - brain ( P21 - 27 ) , as revealed by immunoblotting of crude synaptosomal ( P2 ) , synaptic plasma membrane ( SPM ) , and PSD ( PSD II ) fractions . Analysis of immunoblots reveal significant decrease of IL1RAPL1 in the SPM and PSD fractions of Ptprd - / - brain samples . ( n = 3 mice [ WT and KO ] for each fraction [ P2 , SPM , and PSD ] , mean ± SEM , * * p < 0 . 01 , Student ' s t - test ) .

O O O O B-MEASURED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Decreased levels of synaptic IL1RAPL1 protein in the Ptprd-/- brain (P21-27), as revealed by immunoblotting of crude synaptosomal (P2), synaptic plasma membrane (SPM), and PSD (PSD II) fractions. Analysis of immunoblots reveal significant decrease of IL1RAPL1 in the SPM and PSD fractions of Ptprd-/- brain samples. (n = 3 mice [WT and KO] for each fraction [P2, SPM, and PSD], mean ± SEM, **p<0.01, Student's t-test).


Normal levels of the PTPδ protein in whole - brain lysates of Ptprd - meA - / - mice ( P21 - 27 ) , which contrasts with the Ptprd - / - brain , where PTPδ protein is undetectable .

O O O O B-MEASURED_VAR O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-MEASURED_VAR O O O O

Normal levels of the PTPδ protein in whole-brain lysates of Ptprd-meA-/- mice (P21-27), which contrasts with the Ptprd-/- brain, where PTPδ protein is undetectable.


Strongly decreased levels of tyrosine - phosphorylated PTPδ protein in whole - brain lysates of Ptprd - meA - / - mice ( P21 - 27 ) , as shown by the immunoprecipitation of IL1RAPL1 protein from whole - brain lysates followed by immunoblotting for total and tyrosine - phosphorylated PTPδ protein .

O O O O O O O B-MEASURED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR O O

Strongly decreased levels of tyrosine-phosphorylated PTPδ protein in whole-brain lysates of Ptprd-meA-/- mice (P21-27), as shown by the immunoprecipitation of IL1RAPL1 protein from whole-brain lysates followed by immunoblotting for total and tyrosine-phosphorylated PTPδ protein.


Decreased levels of synaptic IL1RAPL1 protein in the Ptprd - meA - / - brain ( P21 - 27 ) , as revealed by immunoblotting P2 , SPM , and PSD ( PSD II ) fractions . Analysis of immunoblots reveal significant decrease of IL1RAPL1 in the SPM and PSD fractions of Ptprd - meA - / - brain samples . ( n = 3 mice [ Ptprd - meA + / + and Ptprd - meA - / - ] and [ P2 , SPM , and PSD ] , mean ± SEM , * p < 0 . 05 , * * * p < 0 . 001 , Student ' s t - test ) . Note that , while unchanged in the P2 and SPM fractions , PTPδ is significantly decreased in the PSD of Ptprd - meA - / - samples .

O O O O B-MEASURED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O

Decreased levels of synaptic IL1RAPL1 protein in the Ptprd-meA-/- brain (P21-27), as revealed by immunoblotting P2, SPM, and PSD (PSD II) fractions. Analysis of immunoblots reveal significant decrease of IL1RAPL1 in the SPM and PSD fractions of Ptprd-meA-/- brain samples. (n = 3 mice [Ptprd-meA+/+ and Ptprd-meA-/-] and [P2, SPM, and PSD], mean ± SEM, *p<0.05, ***p < 0.001, Student's t-test). Note that, while unchanged in the P2 and SPM fractions, PTPδ is significantly decreased in the PSD of Ptprd-meA-/- samples.


Decreased basal excitatory synaptic transmission in the hippocampal SLM region of Ptprd - meA - / - mice , as shown by the I / O ratio at the TA - SLM pathway ( P20 - 24 ) . ( n = 15 slice from 5 mice [ Ptprd - meA + / + ] , 12 , 4 [ Ptprd - meA - / - ] , mean ± SEM , * p < 0 . 05 ; * * * p < 0 . 001 , two - way RM ANOVA with Holm - Sidak test ) .

O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Decreased basal excitatory synaptic transmission in the hippocampal SLM region of Ptprd-meA-/- mice, as shown by the I/O ratio at the TA-SLM pathway (P20-24). (n = 15 slice from 5 mice [Ptprd-meA+/+], 12, 4 [Ptprd-meA-/-], mean ± SEM, *p < 0.05; ***p<0.001, two-way RM ANOVA with Holm-Sidak test).


Normal presynaptic release in the hippocampal SLM region of Ptprd - meA - / - mice , as shown by the PPF ratio at the TA - SLM pathway ( P20 - 24 ) . ( n = 15 slice from 5 mice [ Ptprd - meA + / + ] and 13 , 4 [ Ptprd - meA - / - ] , mean ± SEM , ns , not significant , two - way RM ANOVA ) .

O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O

Normal presynaptic release in the hippocampal SLM region of Ptprd-meA-/- mice, as shown by the PPF ratio at the TA-SLM pathway (P20-24). (n = 15 slice from 5 mice [Ptprd-meA+/+] and 13, 4 [Ptprd-meA-/-], mean ± SEM, ns, not significant, two-way RM ANOVA).


Hyperactivity of Ptprd - / - mice ( 2 . 5 - 4 months ) in Laboras cages observed during light - off and light - on periods were strongly mimicked by Emx1 - cKO mice , but more weakly by Viaat - cKO mice and not at all by Camk2a - cKO mice . Note also that immobility , a measure that better reflects sleep behavior , showed similar changes among different mouse lines . ( n = 14 mice [ WT ] , 14 [ global KO ] , 16 [ Emx1 - cWT ] , 16 [ Emx1 - cKO ] , 11 [ Viaat - cWT ] , 13 [ Viaat - cKO ] , 18 [ Camk2a - cWT ] and 13 [ Camk2a - cKO ] , mean ± SEM , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ; ns , not significant , two - way RM ANOVA with Holm - Sidak test ) .

O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Hyperactivity of Ptprd-/- mice (2.5-4 months) in Laboras cages observed during light-off and light-on periods were strongly mimicked by Emx1-cKO mice, but more weakly by Viaat-cKO mice and not at all by Camk2a-cKO mice. Note also that immobility, a measure that better reflects sleep behavior, showed similar changes among different mouse lines. (n = 14 mice [WT], 14 [global KO], 16 [Emx1-cWT], 16 [Emx1-cKO], 11 [Viaat-cWT], 13 [Viaat-cKO], 18 [Camk2a-cWT] and 13 [Camk2a-cKO], mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001; ns, not significant, two-way RM ANOVA with Holm-Sidak test).


Hyperactive behavior and decreased immobility of Ptprd - meA - / - mice ( 2 . 5 - 4 months ) in Laboras cages observed during light - off periods and light - on periods , similar to Ptprd - / - mice . ( n = 7 mice for Ptprd - meA + / + and 7 mice for Ptprd - meA - / - , mean ± SEM , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , ns , not significant , two - way RM ANOVA with Holm - Sidak test ) .

O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Hyperactive behavior and decreased immobility of Ptprd-meA-/- mice (2.5-4 months) in Laboras cages observed during light-off periods and light-on periods, similar to Ptprd-/- mice. (n = 7 mice for Ptprd-meA+/+ and 7 mice for Ptprd-meA-/-, mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001, ns, not significant, two-way RM ANOVA with Holm-Sidak test).


Increased WAKE duration and decreased NREM duration during the first three hours of the light - on period in Emx1 - Cre ; Ptprdfl / fl mice ( 2 . 5 - 4 months ) . ( n = 9 mice [ Emx1 - cWT ] and 9 mice [ Emx1 - cKO ] , mean ± SEM , * p < 0 . 05 , * * p < 0 . 01 , ns , not significant , two - way RM ANOVA with Holm - Sidak test ) .

O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Increased WAKE duration and decreased NREM duration during the first three hours of the light-on period in Emx1-Cre;Ptprdfl/fl mice (2.5-4 months). (n = 9 mice [Emx1-cWT] and 9 mice [Emx1-cKO], mean ± SEM, *p < 0.05, **p < 0.01, ns, not significant, two-way RM ANOVA with Holm-Sidak test).


Decreased delta power ( normalized to total power ) in NREM , but not WAKE , oscillations in Emx1 - Cre ; Ptprdfl / fl mice ( 2 . 5 - 4 months ) . Note also the normal theta power ( normalized to total power ) in REM oscillations in Emx1 - Cre ; Ptprdfl / fl mice . ( n = 9 [ Emx1 - cWT ] and 9 [ Emx1 - cKO ] , mean ± SEM , * p < 0 . 05 , * * p < 0 . 01 , ns , not significant , two - way RM ANOVA with Holm - Sidak test ) .

O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Decreased delta power (normalized to total power) in NREM, but not WAKE, oscillations in Emx1-Cre;Ptprdfl/fl mice (2.5-4 months). Note also the normal theta power (normalized to total power) in REM oscillations in Emx1-Cre;Ptprdfl/fl mice. (n = 9 [Emx1-cWT] and 9 [Emx1-cKO], mean ± SEM, *p < 0.05, **p < 0.01, ns, not significant, two-way RM ANOVA with Holm-Sidak test).


( D ) Increased WAKE duration and decreased NREM duration during the first three hours of the light - on period and decreased REM duration in the first half of the light - on period in Ptprd - meA - / - mice ( 2 . 5 - 4 months ) . Note also that Ptprd - meA - / - mice also display increased WAKE duration and decreased NREM duration during the light - off period . ( n = 8 [ Ptprd - meA + / + ] and 8 [ Ptprd - meA - / - ] , mean ± SEM , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , ns , not significant , two - way RM ANOVA with Holm - Sidak test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Increased WAKE duration and decreased NREM duration during the first three hours of the light-on period and decreased REM duration in the first half of the light-on period in Ptprd-meA-/- mice (2.5-4 months). Note also that Ptprd-meA-/- mice also display increased WAKE duration and decreased NREM duration during the light-off period. (n = 8 [Ptprd-meA+/+] and 8 [Ptprd-meA-/-], mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001, ns, not significant, two-way RM ANOVA with Holm-Sidak test).


Decreased delta power ( normalized to total power ) in NREM ( both light - off and light - on periods ) and WAKE ( light - on period ) oscillations , and decreased theta power in REM ( mainly light - on period ) oscillations in Ptprd - meA - / - mice ( 2 . 5 - 4 months ) . ( n = 8 [ Ptprd - meA + / + ] and 8 [ Ptprd - meA - / - ] , mean ± SEM , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , ns , not significant , two - way RM ANOVA with Holm - Sidak test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Decreased delta power (normalized to total power) in NREM (both light-off and light-on periods) and WAKE (light-on period) oscillations, and decreased theta power in REM (mainly light-on period) oscillations in Ptprd-meA-/- mice (2.5-4 months). (n = 8 [Ptprd-meA+/+] and 8 [Ptprd-meA-/-], mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001, ns, not significant, two-way RM ANOVA with Holm-Sidak test).


WT C57BL / 6J mice were injected with 12 . 5 mg / kg LPS , with or without pre - treatment with 10 mg / kg DEX 30 minutes before LPS . Lethality was followed - up . LPS - treated mice ( n = 5 ) are depicted as black circles and DEX / LPS - treated mice ( n = 5 ) are depicted as white circles . P - values for survival curves were analyzed with a Fisher ' s exact test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

WT C57BL/6J mice were injected with 12.5 mg/kg LPS, with or without pre-treatment with 10 mg/kg DEX 30 minutes before LPS. Lethality was followed-up. LPS-treated mice (n=5) are depicted as black circles and DEX/LPS-treated mice (n=5) are depicted as white circles. P-values for survival curves were analyzed with a Fisher's exact test.


WT C57BL / 6J mice were injected with 12 . 5 mg / kg LPS , with or without pre - treatment with 10 mg / kg DEX 30 minutes before LPS . Blood samples were taken 2 hours after LPS and TNF levels were measured in serum ( n = 4 - 5 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O

WT C57BL/6J mice were injected with 12.5 mg/kg LPS, with or without pre-treatment with 10 mg/kg DEX 30 minutes before LPS. Blood samples were taken 2 hours after LPS and TNF levels were measured in serum (n=4-5).


WT C57BL / 6J mice were injected with 12 . 5 mg / kg LPS , with or without pre - treatment with 10 mg / kg DEX 30 minutes before LPS . Three hours after LPS challenge , dextran labeled with FITC was administered by oral gavage . Five hours after gavaging , blood plasma was collected and fluorescence was measured as a degree of intestinal permeability ( λexc / λem = 488 / 520 nm ) ( PBS : n = 3 , LPS and DEX / LPS : n = 5 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

WT C57BL/6J mice were injected with 12.5 mg/kg LPS, with or without pre-treatment with 10 mg/kg DEX 30 minutes before LPS. Three hours after LPS challenge, dextran labeled with FITC was administered by oral gavage. Five hours after gavaging, blood plasma was collected and fluorescence was measured as a degree of intestinal permeability (λexc/λem = 488/520 nm) (PBS: n=3, LPS and DEX/LPS: n=5).


RNA sequencing ( RNAseq ) results of IECs of WT mice ( n = 3 ) treated with PBS or 12 . 5 mg / kg LPS for 6 hours . Heatmap with selection of LPS - induced genes . Color key shows the log2 of normalized counts .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

RNA sequencing (RNAseq) results of IECs of WT mice (n=3) treated with PBS or 12.5 mg/kg LPS for 6 hours. Heatmap with selection of LPS-induced genes. Color key shows the log2 of normalized counts.


WT mice ( n = 5 ) were treated with PBS or 12 . 5 mg / kg LPS with or without 10 mg / kg DEX pre - treatment . Ileum was isolated and gene expression was measured via qPCR .

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WT mice (n=5) were treated with PBS or 12.5 mg/kg LPS with or without 10 mg/kg DEX pre-treatment. Ileum was isolated and gene expression was measured via qPCR.


WT mice ( n = 3 - 5 ) were treated with PBS or 12 . 5 mg / kg LPS with or without 10 mg / kg DEX pre - treatment . Ileum was isolated 6 hours after LPS challenge . ( A ) p - MLKL and MLKL protein levels and ( B ) RIPK3 protein levels were analyzed via western blot using ACTIN as a loading control . Expression levels were quantified using FIJI and normalized to ACTIN levels . P - values were calculated using 1 - way ANOVA .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

WT mice (n=3-5) were treated with PBS or 12.5 mg/kg LPS with or without 10 mg/kg DEX pre-treatment. Ileum was isolated 6 hours after LPS challenge. (A) p-MLKL and MLKL protein levels and (B) RIPK3 protein levels were analyzed via western blot using ACTIN as a loading control. Expression levels were quantified using FIJI and normalized to ACTIN levels. P-values were calculated using 1-way ANOVA.


Gene expression ( Defa6 , lysozyme ) was measured via qPCR . P - values were calculated using 1 - way ANOVA . All bars represent mean ± SEM . * * * * P < 0 . 0001 ; * * * P < 0 . 001 ; * * P ≤ 0 . 01 ; * P ≤ 0 . 05 .

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Gene expression (Defa6, lysozyme) was measured via qPCR. P-values were calculated using 1-way ANOVA. All bars represent mean ± SEM. ****P< 0.0001; *** P<0.001; ** P≤ 0.01; * P≤ 0.05.


H & E staining was performed on ileum samples . Scale bar : 50µm .

H&E staining was performed on ileum samples. Scale bar: 50µm.


Lysozyme expression ( in red ) was detected via IHC , with Hoechst counterstaining . Scale bars : 50µm . Quantification of lysozyme expression . For every picture ( n = 3 - 4 per treatment ) , a selection of 10 crypts was made . Lysozyme positive signal was quantified in this region of interest ( ROI ) using an automated script in QuPath . Values are the positive area relative to total area of ROI .

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Lysozyme expression (in red) was detected via IHC, with Hoechst counterstaining. Scale bars: 50µm. Quantification of lysozyme expression. For every picture (n=3-4 per treatment), a selection of 10 crypts was made. Lysozyme positive signal was quantified in this region of interest (ROI) using an automated script in QuPath. Values are the positive area relative to total area of ROI.


TNFR1 + / + ( n = 9 ) and TNFR1 - / - mice ( n = 11 ) were treated with 12 . 5 mg / kg LPS and lethality was monitored . P - values were analyzed with a Fisher ' s exact test .

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TNFR1+/+ (n=9) and TNFR1-/- mice (n=11) were treated with 12.5 mg/kg LPS and lethality was monitored. P-values were analyzed with a Fisher's exact test.


TLR4fl / fl mice ( n = 7 ) and TLR4VillinKO ( n = 9 ) mice were treated with 12 . 5 mg / kg LPS and lethality was monitored . P - values were analyzed with a Fisher ' s exact test .

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TLR4fl/fl mice (n=7) and TLR4VillinKO (n=9) mice were treated with 12.5 mg/kg LPS and lethality was monitored. P-values were analyzed with a Fisher's exact test.


TNFR1fl / fl mice ( n = 13 ) and TNFR1VillinKO ( n = 10 ) mice were treated with 12 . 5 mg / kg LPS and lethality was monitored . P - values were analyzed with a Fisher ' s exact test .

TNFR1fl/fl mice (n=13) and TNFR1VillinKO (n=10) mice were treated with 12.5 mg/kg LPS and lethality was monitored. P-values were analyzed with a Fisher's exact test.


TNFR1 + / + and TNFR1 - / - mice ( n = 4 - 6 ) were treated with 12 . 5 mg / kg LPS . Ileum was isolated and gene expression was measured via qPCR . Fold changes are shown on the graph .

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TNFR1+/+ and TNFR1-/- mice (n=4-6) were treated with 12.5 mg/kg LPS. Ileum was isolated and gene expression was measured via qPCR. Fold changes are shown on the graph.


TNFR1fl / fl and TNFR1VillinKO mice ( n = 4 - 6 ) were treated with 12 . 5 mg / kg LPS . Ileum was isolated and gene expression was measured via qPCR . Fold changes are shown on the graph .

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TNFR1fl/fl and TNFR1VillinKO mice (n=4-6) were treated with 12.5 mg/kg LPS. Ileum was isolated and gene expression was measured via qPCR. Fold changes are shown on the graph.


GRfl / fl ( n = 8 , ) and GRLysMKO mice ( n = 4 ) were treated with 2 . 5 mg / kg LPS and lethality was monitored . P - values were analyzed with a Fisher ' s exact test .

GRfl/fl (n=8,) and GRLysMKO mice (n=4) were treated with 2.5 mg/kg LPS and lethality was monitored. P-values were analyzed with a Fisher's exact test.


GRLysMKO mice were treated with PBS ( n = 12 ) or 10 mg / kg DEX ( n = 13 ) before a lethal dose of LPS and lethality was monitored . P - values were analyzed with a Fisher ' s exact test .

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GRLysMKO mice were treated with PBS (n=12) or 10 mg/kg DEX (n=13) before a lethal dose of LPS and lethality was monitored. P-values were analyzed with a Fisher's exact test.


TNF measurement in the serum of GRfl / fl ( n = 4 ) and GRLysMKO mice ( n = 3 ) . Mice were treated with 2 . 5 mg / kg LPS and blood was drawn 2 hours later . TNF concentration was measured with ELISA .

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TNF measurement in the serum of GRfl/fl (n=4) and GRLysMKO mice (n=3). Mice were treated with 2.5 mg/kg LPS and blood was drawn 2 hours later. TNF concentration was measured with ELISA.


TNF measurement in the serum of GRfl / fl and GRLysMKO ( n = 4 ) . Mice were treated with PBS or a lethal dose of LPS ( 12 . 5 and 2 . 5 mg / kg for GRfl / fl and GRLysMKO mice respectively ) , with or without pre - treatment with 10 mg / kg DEX and blood was drawn 2 hours later . TNF concentration was measured with ELISA .

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TNF measurement in the serum of GRfl/fl and GRLysMKO (n=4). Mice were treated with PBS or a lethal dose of LPS (12.5 and 2.5 mg/kg for GRfl/fl and GRLysMKO mice respectively) , with or without pre-treatment with 10 mg/kg DEX and blood was drawn 2 hours later. TNF concentration was measured with ELISA.


GRfl / fl ( n = 4 ) and GRLysMKO mice ( n = 3 - 4 ) were treated with 2 . 5 mg / kg LPS . Ileum was isolated after 6 hours and gene expression was measured via qPCR . Fold changes are depicted on the graphs .

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GRfl/fl (n=4) and GRLysMKO mice (n=3-4) were treated with 2.5 mg/kg LPS. Ileum was isolated after 6 hours and gene expression was measured via qPCR. Fold changes are depicted on the graphs.


GRfl / fl and GRLysMKO mice ( n = 4 ) were treated with a PBS or LPS ( 12 . 5 and 2 . 5 mg / kg for GRfl / fl and GRLysMKO mice respectively ) with or without 10 mg / kg DEX pre - treatment of 30 minutes . Ileum was isolated after 6 hours and gene expression was measured via qPCR . Fold changes are depicted on the graphs .

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GRfl/fl and GRLysMKO mice (n=4) were treated with a PBS or LPS (12.5 and 2.5 mg/kg for GRfl/fl and GRLysMKO mice respectively) with or without 10 mg/kg DEX pre-treatment of 30 minutes. Ileum was isolated after 6 hours and gene expression was measured via qPCR. Fold changes are depicted on the graphs.


GRfl / fl ( n = 8 ) and GRVillinKO mice ( n = 10 ) were treated with 5 mg / kg LPS and lethality was monitored . P - values were analyzed with a Fisher ' s exact test .

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GRfl/fl (n=8) and GRVillinKO mice (n=10) were treated with 5 mg/kg LPS and lethality was monitored. P-values were analyzed with a Fisher's exact test.


GRVillinKO mice were treated with PBS ( n = 9 ) or 10 mg / kg DEX ( n = 10 ) before 5 mg / kg LPS and lethality was monitored . P - values were analyzed with a Fisher ' s exact test .

GRVillinKO mice were treated with PBS (n=9) or 10 mg/kg DEX (n=10) before 5 mg/kg LPS and lethality was monitored. P-values were analyzed with a Fisher's exact test.


TNF measurement in the serum of GRfl / fl ( n = 4 ) and GRVillinKO mice ( n = 4 ) . Mice were treated with 5 mg / kg LPS and blood was drawn 2 hours later .

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TNF measurement in the serum of GRfl/fl (n=4) and GRVillinKO mice (n= 4). Mice were treated with 5 mg/kg LPS and blood was drawn 2 hours later.


TNF measurement in the serum of GRfl / fl ( n = 4 ) and GRVillinKO mice ( n = 4 ) . Mice were treated with PBS or LPS ( 12 . 5 and 5 mg / kg for GRfl / fl and GRVillinKO mice respectively ) with or without pre - treatment with 10 mg / kg DEX for 30 minutes . Blood was taken 2 hours after LPS challenge and TNF concentrations were measured with ELISA .

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TNF measurement in the serum of GRfl/fl (n=4) and GRVillinKO mice (n=4). Mice were treated with PBS or LPS (12.5 and 5 mg/kg for GRfl/fl and GRVillinKO mice respectively) with or without pre-treatment with 10 mg/kg DEX for 30 minutes. Blood was taken 2 hours after LPS challenge and TNF concentrations were measured with ELISA.


GRVillinKO mice were treated with PBS ( n = 8 ) or a neutralizing P55 ( TNFR1 ) antibody ( n = 9 ) 2 hours before and 8 hours after 5 mg / kg LPS and lethality was monitored . P - values were analyzed with a Fisher ' s exact test .

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GRVillinKO mice were treated with PBS (n=8) or a neutralizing P55 (TNFR1) antibody (n=9) 2 hours before and 8 hours after 5 mg/kg LPS and lethality was monitored. P-values were analyzed with a Fisher's exact test.


Ileum was isolated from non - stimulated GRfl / fl ( n = 4 ) and GRVillinKO mice ( n = 4 ) and gene expression was measured via qPCR . Expression levels are shown as fold induction with GRfl / fl expression values set at 1 . P - values were calculated using a student ' s t - test .

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Ileum was isolated from non-stimulated GRfl/fl (n=4) and GRVillinKO mice (n=4) and gene expression was measured via qPCR. Expression levels are shown as fold induction with GRfl/fl expression values set at 1. P-values were calculated using a student's t-test.


GRfl / fl ( n = 4 ) and GRVillinKO mice ( n = 4 ) were treated with 5 mg / kg LPS . Ileum was isolated and gene expression was measured via qPCR .

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GRfl/fl (n=4) and GRVillinKO mice (n=4) were treated with 5 mg/kg LPS. Ileum was isolated and gene expression was measured via qPCR.


Left : LPS dose - response curves of GRwt / wt ( n = 3 - 7 per LPS dose ) and GRdim / dim mice ( n = 3 - 7 per LPS dose ) treated with 1 . 5 mg / kg LPS . Right : LD50 values of LPS are depicted on top of each bar for each group .

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Left: LPS dose-response curves of GRwt/wt (n=3-7 per LPS dose) and GRdim/dim mice (n=3-7 per LPS dose) treated with 1.5 mg/kg LPS. Right: LD50 values of LPS are depicted on top of each bar for each group.


GRdim / dim mice were treated with PBS ( n = 5 ) or 10 mg / kg DEX ( n = 5 ) before 1 . 5 mg / kg LPS and survival was monitored . P - values were analyzed with a Fisher ' s exact test .

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GRdim/dim mice were treated with PBS (n=5) or 10 mg/kg DEX (n=5) before 1.5 mg/kg LPS and survival was monitored. P-values were analyzed with a Fisher's exact test.


TNF measurement in the serum of GRwt / wt ( n = 5 ) and GRdim / dim mice ( n = 5 ) . Mice were treated with 1 . 5 mg / kg LPS and blood was drawn 2 hours later . TNF concentration was measured with ELISA .

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TNF measurement in the serum of GRwt/wt (n=5) and GRdim/dim mice (n= 5). Mice were treated with 1.5 mg/kg LPS and blood was drawn 2 hours later. TNF concentration was measured with ELISA.


TNF measurement in the serum of GRwt / wt and GRdim / dim mice ( n = 4 ) treated with PBS or LPS ( 12 . 5 and 1 . 5 mg / kg in GRwt / wt and GRdim / dim mice respectively ) with or without pretreatment with 10 mg / kg DEX . Blood was taken 2 hours after LPS challenge and TNF concentrations were measured with ELISA .

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TNF measurement in the serum of GRwt/wt and GRdim/dim mice (n=4) treated with PBS or LPS (12.5 and 1.5 mg/kg in GRwt/wt and GRdim/dim mice respectively) with or without pretreatment with 10 mg/kg DEX. Blood was taken 2 hours after LPS challenge and TNF concentrations were measured with ELISA.


GRdim / dim mice were treated with PBS ( n = 6 ) or a neutralizing P55 ( TNFR1 ) antibody ( n = 8 ) 2 hours before and 8 hours after 1 . 5 mg / kg LPS and lethality was monitored . P - values were analyzed with a Fisher ' s exact test .

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GRdim/dim mice were treated with PBS (n=6) or a neutralizing P55 (TNFR1) antibody (n=8) 2 hours before and 8 hours after 1.5 mg/kg LPS and lethality was monitored. P-values were analyzed with a Fisher's exact test.


Tofacitinib ( n = 6 ) or vehicle ( n = 7 ) was given orally to GRdim / dim mice . After 1 . 5 mg / kg LPS injection survival was monitored and p - values were analyzed with a Fisher ' s exact test .

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Tofacitinib (n=6) or vehicle (n=7) was given orally to GRdim/dim mice. After 1.5 mg/kg LPS injection survival was monitored and p-values were analyzed with a Fisher's exact test.


GRwt / wt ( n = 4 ) and GRdim / dim ( n = 4 ) mice were treated with 1 . 5 mg / kg LPS . Ileum was isolated and gene expression was measured via qPCR . Fold changes are depicted on the graphs .

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GRwt/wt (n=4) and GRdim/dim (n=4) mice were treated with 1.5 mg/kg LPS. Ileum was isolated and gene expression was measured via qPCR. Fold changes are depicted on the graphs.


GRdim / dim mice ( n = 4 ) were treated with PBS or 1 . 5 mg / kg LPS with or without 10 mg / kg DEX pretreatment . Ileum was isolated and gene expression was measured via qPCR . Fold changes are depicted on the graphs .

GRdim/dim mice (n=4) were treated with PBS or 1.5 mg/kg LPS with or without 10 mg/kg DEX pretreatment. Ileum was isolated and gene expression was measured via qPCR. Fold changes are depicted on the graphs.


For SPR measurements , IL - 27Rα or GP130 were immobilized on the chip surface by biotin - streptavidin interaction and IL - 27 were flowed across the chip in solution . SPR data are representative of three biological replicates . Kinetic charts for IL - 27Rα ( A ) and GP130 ( B ) . Concentrations used are shown on the curves . Data traces were fitted using a 1 : 1 interaction model ( black ) to quantify the kinetics ( kon , koff ) and binding affinity ( KD ) of the interactions .

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For SPR measurements, IL-27Rα or GP130 were immobilized on the chip surface by biotin-streptavidin interaction and IL-27 were flowed across the chip in solution. SPR data are representative of three biological replicates. Kinetic charts for IL-27Rα (A) and GP130 (B). Concentrations used are shown on the curves. Data traces were fitted using a 1:1 interaction model (black) to quantify the kinetics (kon, koff) and binding affinity (KD) of the interactions.


For SPR measurements , IL - 27Rα were immobilized on the chip surface by biotin - streptavidin interaction and p28 were flowed across the chip in solution . SPR data are representative of three biological replicates . ( C , Equilibrium chart for p28 binding to IL - 27Rα ( C , left panel ) and curve - fitting to data points generated at various concentrations of p28 ( C , right panel ) .

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For SPR measurements, IL-27Rα were immobilized on the chip surface by biotin-streptavidin interaction and p28 were flowed across the chip in solution. SPR data are representative of three biological replicates. (C, Equilibrium chart for p28 binding to IL-27Rα (C, left panel) and curve-fitting to data points generated at various concentrations of p28 (C, right panel).


For SPR measurements , GP130 were immobilized on the chip surface by biotin - streptavidin interaction and p28 were flowed across the chip in solution . SPR data are representative of three biological replicates . ( D ) Equilibrium chart for p28 binding GP130 .

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For SPR measurements, GP130 were immobilized on the chip surface by biotin-streptavidin interaction and p28 were flowed across the chip in solution. SPR data are representative of three biological replicates. (D) Equilibrium chart for p28 binding GP130.


( F , G ) Dose response curves for pSTAT1 ( F ) and pSTAT3 ( G ) in resting mouse CD8 + T cells . Cells were stimulated with mIL - 27 or p28 for 15 minutes with the indicated doses . Data shown are the mean of four biological replicates with error bars depicting standard error of the mean .

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(F,G) Dose response curves for pSTAT1 (F) and pSTAT3 (G) in resting mouse CD8+ T cells. Cells were stimulated with mIL-27 or p28 for 15 minutes with the indicated doses. Data shown are the mean of four biological replicates with error bars depicting standard error of the mean.


( B - D ) Flow cytometric quantification of DHE > Eth . positive wild type yeast cells ( allowing the detection of both dead cells and cells exhibiting sub - lethal oxidative stress ) after 42 h ( B ) or 66 h ( C , D ) expressing Abeta constructs . Dot plots show all data points along with the mean ( bar ) ± s . d . . n = 5 - 12 biologically independent cultures . * * * , p < 0 . 001 , * * , p < 0 . 01 , * , p < 0 . 05 . ANOVA with Tukey ' s post - hoc test .

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(B-D) Flow cytometric quantification of DHE>Eth. positive wild type yeast cells (allowing the detection of both dead cells and cells exhibiting sub-lethal oxidative stress) after 42 h (B) or 66 h (C, D) expressing Abeta constructs. Dot plots show all data points along with the mean (bar) ± s.d.. n = 5-12 biologically independent cultures. ***, p < 0.001, **, p < 0.01, *, p < 0.05. ANOVA with Tukey's post-hoc test.


( E ) Annexin V ( AV ) / PI co - staining to assess cell death in yeast expressing A42 after 48 h . Data represent means ± s . d . n = 6 biologically independent cultures . * * * , p < 0 . 001 ( comparing PI positive populations ) . Unpaired , two - tailed t - test .

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(E) Annexin V (AV)/PI co-staining to assess cell death in yeast expressing A42 after 48 h. Data represent means ± s.d. n = 6 biologically independent cultures. ***, p < 0.001 (comparing PI positive populations). Unpaired, two-tailed t-test.


( F ) Immunoblot of whole cell extract ( WCE ) of wild type yeast cells after 16 h expression of Abeta constructs . For western blot ( WB ) Abeta - specific antibody ( Abeta ) 6E10 was used showing long ( Long exp . ) and short time exposure ( Short exp . ) . Ssc1 was used as a loading control .

(F) Immunoblot of whole cell extract (WCE) of wild type yeast cells after 16 h expression of Abeta constructs. For western blot (WB) Abeta-specific antibody (Abeta) 6E10 was used showing long (Long exp.) and short time exposure (Short exp.). Ssc1 was used as a loading control.


( G ) Confocal microscopy of wild type yeast cells after 18 h of Abeta expression . Colors indicate fluorescence intensity .

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(G) Confocal microscopy of wild type yeast cells after 18 h of Abeta expression. Colors indicate fluorescence intensity.


( A ) Immunoblot of total cytoplasmic post - nuclear supernatant ( PNS ) , mitochondrial , microsomal and cytosolic fractions of wild type yeast cells after 18 h expression of EGFP - Abeta42 ( A42 ) , EGFP - Abeta40 ( A40 ) and EGFP empty vector ( ev ) using Abeta - specific antibody ( Abeta ) 6E10 with long ( Long exp . ) and short time exposure ( Short exp . ) . Purity of fractions was tested with antibodies against Cyc1 ( mitochondria ) , Sec61 ( microsomes ) and Pgk1 ( cytosol ) .

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(A) Immunoblot of total cytoplasmic post-nuclear supernatant (PNS), mitochondrial, microsomal and cytosolic fractions of wild type yeast cells after 18 h expression of EGFP-Abeta42 (A42), EGFP-Abeta40 (A40) and EGFP empty vector (ev) using Abeta-specific antibody (Abeta) 6E10 with long (Long exp.) and short time exposure (Short exp.). Purity of fractions was tested with antibodies against Cyc1 (mitochondria), Sec61 (microsomes) and Pgk1 (cytosol).


( B ) Cellular ATP content of wild type yeast cells after 42 h expression of EGFP - A42 ( A42 ) or EGFP empty vector ( ev ) . Dot plots show all data points along with the mean ( bar ) ± s . d . . n = 16 biologically independent cultures . * * , p < 0 . 01 . Unpaired , two - tailed t - test .

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(B) Cellular ATP content of wild type yeast cells after 42 h expression of EGFP-A42 (A42) or EGFP empty vector (ev). Dot plots show all data points along with the mean (bar) ± s.d.. n = 16 biologically independent cultures. **, p < 0.01. Unpaired, two-tailed t-test.