A . HPLC - based PG digestion assay representing EPase activity . The graph shows the relative percentage of the muropeptide TetraTetra present at the end of the incubation period for each protein as described in materials and methods . MepM and PBP4 were incubated with sacculi , whilst MepS and PBP7 were incubated with soluble muropeptides , both from E . coli MC1061 , respectively . Values are mean ± standard deviation of three independent experiments . To calculate significance , the data were fit using a linear model . Calculated means were compared using Tukey ' s HSD test , resulting in p - values corrected for multiple testing . Relevant p - values are highlighted directly in the figure ( * < 0 . 05 ; * * < 0 . 01 , * * * < 0 . 001 )

O O B-EXP_ASSAY O O B-SUBCELLULAR B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-SUBCELLULAR O O B-GENEPROD O B-GENEPROD O O O O B-SMALL_MOLECULE O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. HPLC-based PG digestion assay representing EPase activity. The graph shows the relative percentage of the muropeptide TetraTetra present at the end of the incubation period for each protein as described in materials and methods. MepM and PBP4 were incubated with sacculi, whilst MepS and PBP7 were incubated with soluble muropeptides, both from E. coli MC1061, respectively. Values are mean ± standard deviation of three independent experiments. To calculate significance, the data were fit using a linear model. Calculated means were compared using Tukey's HSD test, resulting in p-values corrected for multiple testing. Relevant p-values are highlighted directly in the figure (*<0.05; **<0.01, ***<0.001)


B . Genetic interactions of nlpI with EPase genes . Strains were arrayed using a Rotor HDA replicator on Lennox LB agar plates and incubated for 12 h at 37 ° C . Each plate contained 384 colonies , 96 from the wildtype , single mutants and double mutants . An example of a 384 plate is shown . Double mutants were made twice , swapping the resistance markers to the two single mutants . Colony integral opacity was quantified as a fitness readout , using the image analysis software Iris ( Kritikos et al . , 2017 ) . Bar plots shows the averaged values of 2 biological experiments , each having 96 technical replicates ( i = 2 , n = 192 ) . The error bars represent the 95 % confidence interval .

O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O 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. Genetic interactions of nlpI with EPase genes. Strains were arrayed using a Rotor HDA replicator on Lennox LB agar plates and incubated for 12 h at 37°C. Each plate contained 384 colonies, 96 from the wildtype, single mutants and double mutants. An example of a 384 plate is shown. Double mutants were made twice, swapping the resistance markers to the two single mutants. Colony integral opacity was quantified as a fitness readout, using the image analysis software Iris (Kritikos et al., 2017). Bar plots shows the averaged values of 2 biological experiments, each having 96 technical replicates (i=2, n=192). The error bars represent the 95% confidence interval.


C . nlpI deletion changes the morphology of EPase - mutant strains . The graph shows the cell width of single and double deletions strains ( 800 < n < 2000 cells ) . The box has a medium between 25 and 75 % . The whiskers with the upper and lower vertical line indicating the 95 and 5 % . The dots are individual points outside the 5 and 95 % range . Above the graph are representative images of cells lacking MepS or PBP4 in combination with a deletion of NlpI . The scale bar equals 2 µm . Gene encoding protein legend : nlpI encodes NlpI , dacB encodes PBP4 , pbpG encode PBP7 , mepM encodes MepM , meps encodes MepS .

O O B-GENEPROD O O O B-EXP_ASSAY O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O

C. nlpI deletion changes the morphology of EPase-mutant strains. The graph shows the cell width of single and double deletions strains (800 < n < 2000 cells). The box has a medium between 25 and 75%. The whiskers with the upper and lower vertical line indicating the 95 and 5%. The dots are individual points outside the 5 and 95% range. Above the graph are representative images of cells lacking MepS or PBP4 in combination with a deletion of NlpI. The scale bar equals 2 µm. Gene encoding protein legend: nlpI encodes NlpI, dacB encodes PBP4, pbpG encode PBP7, mepM encodes MepM, meps encodes MepS.


D . Inducible mepS expression system ( pBAD30 ) strongly overproduces MepS . Strains were grown in LB at 30 ° C and cells were collected at OD600 ≈ 0 . 4 . The level of MepS contained in the membrane fraction was detected using purified anti - MepS antibody .

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D. Inducible mepS expression system (pBAD30) strongly overproduces MepS. Strains were grown in LB at 30°C and cells were collected at OD600≈0.4. The level of MepS contained in the membrane fraction was detected using purified anti-MepS antibody.


E . Visualization of the effect of MepS absence or its overexpression on cell width by phase contrast microscopy . Cultures were grown in LB at 30 ° C and aliquots of culture were taken at OD600 ≈ 0 . 1 . The scale bar equals 5 μm .

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E. Visualization of the effect of MepS absence or its overexpression on cell width by phase contrast microscopy. Cultures were grown in LB at 30°C and aliquots of culture were taken at OD600≈0.1. The scale bar equals 5 μm.


F . MepS level modulates cell width . The graph shows the distribution of mean cell width for each cell , with n corresponding to the number of cells measured for each strain and the median width for the population being indicated by a dotted line and referred to as w .

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F. MepS level modulates cell width. The graph shows the distribution of mean cell width for each cell, with n corresponding to the number of cells measured for each strain and the median width for the population being indicated by a dotted line and referred to as w.


G . Relative fitness of ΔnlpI , ΔmepS and ΔnlpIΔmepS mutants . Strains were arrayed using a Rotor HDA replicator on Lennox LB agar plates supplemented 10 % sucrose , or LB agar plates containing 0 mM or 500 mM NaCl . Plates were incubated for 12 h at 37 ° C . Each plate contained 384 colonies , 96 from the wildtype , single mutants and double mutants .

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G. Relative fitness of ΔnlpI, ΔmepS and ΔnlpIΔmepS mutants. Strains were arrayed using a Rotor HDA replicator on Lennox LB agar plates supplemented 10% sucrose, or LB agar plates containing 0 mM or 500 mM NaCl. Plates were incubated for 12 h at 37°C. Each plate contained 384 colonies, 96 from the wildtype, single mutants and double mutants.


H . Cells of wildtype ( WT ) , ΔnlpI , ΔmepS and ΔnlpIΔmepS containing multicopy plasmids with lacZ were grown onto CPRG indicator agar to assay envelope integrity . CPRG ( yellow ) cannot penetrate intact Gram - negative envelopes . Its conversion by intracellular β - galactosidase to CPR ( red ) indicates loss of envelope integrity .

O O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O O O O O O B-EXP_ASSAY I-EXP_ASSAY O

H. Cells of wildtype (WT), ΔnlpI, ΔmepS and ΔnlpIΔmepS containing multicopy plasmids with lacZ were grown onto CPRG indicator agar to assay envelope integrity. CPRG (yellow) cannot penetrate intact Gram-negative envelopes. Its conversion by intracellular β-galactosidase to CPR (red) indicates loss of envelope integrity.


A . Phase contrast image and corresponding fluorescence SIM image of BW25113 cells that have been grown in LB at 37 ° C and immunolabelled with specific antibodies against NlpI . Scale bar equals 2 µm .

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A. Phase contrast image and corresponding fluorescence SIM image of BW25113 cells that have been grown in LB at 37°C and immunolabelled with specific antibodies against NlpI. Scale bar equals 2 µm.


B . Genetic interactions of NlpI with PG machineries . An example of a 384 plate is shown . Bar plots shows the averaged values of 2 experiments ( i = 2 , n = 192 ) . Error bars denote the 95 % confidence interval of the mean .

O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O 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. Genetic interactions of NlpI with PG machineries. An example of a 384 plate is shown. Bar plots shows the averaged values of 2 experiments (i=2, n=192). Error bars denote the 95% confidence interval of the mean.


C . NlpI deletion exacerbates the morphological defects of the PBP1B / LpoB - mutant strains . The graph shows the cell width of single and double deletions strains ( 800 < n < 2000 cells ) . The box has a medium between 25 and 75 % . The whiskers with the upper and lower vertical line indicating the 95 and 5 % . The dots are individual points outside the 5 and 95 % range . Representative images of the strains are shown above the graph . The scale bar equals 2 µm . Gene encoding protein legend : nlpI encodes NlpI , mrcA encodes PBP1A , lpoA encodes LpoA , mrcB encodes PBP1B , lpoB encodes LpoB .

O O B-GENEPROD O O O B-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O

C. NlpI deletion exacerbates the morphological defects of the PBP1B/LpoB-mutant strains. The graph shows the cell width of single and double deletions strains (800 < n < 2000 cells). The box has a medium between 25 and 75%. The whiskers with the upper and lower vertical line indicating the 95 and 5%. The dots are individual points outside the 5 and 95% range. Representative images of the strains are shown above the graph. The scale bar equals 2 µm. Gene encoding protein legend: nlpI encodes NlpI, mrcA encodes PBP1A, lpoA encodes LpoA, mrcB encodes PBP1B, lpoB encodes LpoB.


D . Dissociation constants for interactions between PBP1A and LpoA with NlpI , MepM , MepS , PBP4 and PBP7 as determined by MST . The values are mean ± standard deviation of three independent experiments . 1PBP1A was used as fluorescently labelled protein in all assays . 2LpoA was used as unlabelled ligand in all combinations , except with MepM and PBP4 .

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D. Dissociation constants for interactions between PBP1A and LpoA with NlpI, MepM, MepS, PBP4 and PBP7 as determined by MST. The values are mean ± standard deviation of three independent experiments. 1PBP1A was used as fluorescently labelled protein in all assays. 2LpoA was used as unlabelled ligand in all combinations, except with MepM and PBP4.


A - B . PBP4 has different interaction sites for PBP1A / LpoA and NlpI as shown by a single concentration MST assay . Plots show the FNorm or fluorescence values of fluorescently labelled PBP1A or LpoA with or without PBP4 , NlpI or PBP4 - NlpI . Values are mean ± standard deviation of three independent experiments .

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A-B. PBP4 has different interaction sites for PBP1A/LpoA and NlpI as shown by a single concentration MST assay. Plots show the FNorm or fluorescence values of fluorescently labelled PBP1A or LpoA with or without PBP4, NlpI or PBP4-NlpI. Values are mean ± standard deviation of three independent experiments.


A . Estimated number of molecules and molarity of PBP1a / LpoA and EPases . 1Numbers obtained by ribosomal profiling in rich growth medium ( Li et al . , 2014 ) . 2Concentration of monomer . 3Decreases in the presence of NlpI ( Singh et al . , 2015 ) . 4 " no change detected " . The periplasmic concentrations of proteins were estimated for a cell with periplasmic volume of 3 . 33 × 10 - 16 L , where 1 molecule corresponds to 5 nM .

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A. Estimated number of molecules and molarity of PBP1a/LpoA and EPases. 1Numbers obtained by ribosomal profiling in rich growth medium (Li et al., 2014). 2Concentration of monomer. 3Decreases in the presence of NlpI (Singh et al., 2015). 4"no change detected". The periplasmic concentrations of proteins were estimated for a cell with periplasmic volume of 3.33 ×10-16 L, where 1 molecule corresponds to 5 nM.


( B ) Islet graft survival of mice receiving vehicle ( PBS ) or IL - 33 was assessed by monitoring blood glucose and calculated using the Kaplan - Meier method . Cumulative data from two independent experiments are shown . Statistical analysis was performed with a log - rank test . * * * P < 0 . 001 vs islet + vehicle . ( C ) Blood glucose level of mice treated with IL - 33 ( the horizontal black line indicates a BGL of 16 mmol / liter , the threshold for rejection ) . Each line represents one mouse .

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(B) Islet graft survival of mice receiving vehicle (PBS) or IL-33 was assessed by monitoring blood glucose and calculated using the Kaplan-Meier method. Cumulative data from two independent experiments are shown. Statistical analysis was performed with a log-rank test. ***P<0.001 vs islet+vehicle. (C) Blood glucose level of mice treated with IL-33 (the horizontal black line indicates a BGL of 16 mmol/liter, the threshold for rejection). Each line represents one mouse.


( D ) Intraperitoneal glucose tolerance test ( IPGTT ) was assessed in normal mice , islet transplant mice receiving vehicle ( on the day when grafts were considered rejected ) , and islet transplant mice treated with IL - 33 ( at day 30 and day 80 post - islet transplantation ) . Data shown are the mean ± SEM ( n = 6 - 9 per group ) . ( E ) Area under the curve ( AUC ) for IPGTT was assessed . Data shown are the mean ± SEM ( n = 6 - 9 per group ) and a one - way ANOVA was performed , * * * P < 0 . 001 .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O B-ORGANISM O B-TISSUE O B-ORGANISM O O O O O O O B-EXP_ASSAY O O O O O O B-TISSUE O B-ORGANISM O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O B-EXP_ASSAY O O O O O O O O O O O O O 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) Intraperitoneal glucose tolerance test (IPGTT) was assessed in normal mice, islet transplant mice receiving vehicle (on the day when grafts were considered rejected), and islet transplant mice treated with IL-33 (at day 30 and day 80 post-islet transplantation). Data shown are the mean ± SEM (n=6-9 per group). (E) Area under the curve (AUC) for IPGTT was assessed. Data shown are the mean ± SEM (n=6-9 per group) and a one-way ANOVA was performed, ***P<0.001.


( F ) Representative immunohistochemical staining for insulin in graft samples from mice receiving vehicle or IL - 33 . Scale bar = 100 µm .

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(F) Representative immunohistochemical staining for insulin in graft samples from mice receiving vehicle or IL-33. Scale bar = 100 µm.


C57BL / 6 mice were treated with mouse recombinant IL - 33 or PBS daily for 5 consecutive days . ( A , B ) Representative FACS analysis showing the proportion of Tregs ( CD4 + Foxp3 + ) in the CD4 + T cell compartment from the spleens ( A ) and kidneys ( B ) at day 3 after treatment in C57BL / 6 mice receiving PBS ( n = 4 ) or IL - 33 ( n = 6 ) .

O O O B-ORGANISM O O O B-ORGANISM O B-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O O B-EXP_ASSAY O O O O O B-CELL_TYPE O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O O B-TISSUE O O O O B-TISSUE O O O O O O O O O O O O B-ORGANISM O B-SMALL_MOLECULE O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O

C57BL/6 mice were treated with mouse recombinant IL-33 or PBS daily for 5 consecutive days. (A, B) Representative FACS analysis showing the proportion of Tregs (CD4+Foxp3+) in the CD4+ T cell compartment from the spleens (A) and kidneys (B) at day 3 after treatment in C57BL/6 mice receiving PBS (n=4) or IL-33 (n=6).


C57BL / 6 mice were treated with mouse recombinant IL - 33 or PBS daily for 5 consecutive days . ( C , D ) Representative FACS analysis showing the proportion of ILC2s ( Lin - GATA - 3 + ) in the CD45 + leukocyte compartment from the spleens ( C ) and kidneys ( D ) at day 3 after treatment in C57BL / 6 mice receiving PBS ( n = 4 ) or IL - 33 ( n = 6 ) .

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C57BL/6 mice were treated with mouse recombinant IL-33 or PBS daily for 5 consecutive days. (C, D) Representative FACS analysis showing the proportion of ILC2s (Lin-GATA-3+) in the CD45+ leukocyte compartment from the spleens (C) and kidneys (D) at day 3 after treatment in C57BL/6 mice receiving PBS (n=4) or IL-33 (n=6).


( E , F ) proportion of Tregs ( E ) and ILC2s ( F ) in the spleen and kidney in PBS - injected controls ( n = 4 ) and at weeks 1 - 12 after IL - 33 treatment ( n = 4 - 6 per IL - 33 - treated group ) . Data information : Data shown are the mean ± SEM ; Statistical analysis was performed with an unpaired t - test , * * P < 0 . 01 , * * * P < 0 . 001 .

O O O O O O O B-CELL_TYPE O O O O O O O O O O B-TISSUE O B-TISSUE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E, F) proportion of Tregs (E) and ILC2s (F) in the spleen and kidney in PBS-injected controls (n=4) and at weeks 1-12 after IL-33 treatment (n=4-6 per IL-33-treated group). Data information: Data shown are the mean ± SEM; Statistical analysis was performed with an unpaired t-test, **P<0.01, ***P<0.001.


( B - D ) Proportion or numbers of CD4 + Foxp3 + Tregs in the spleens , kidneys and islet grafts of normal , islet transplant mice receiving vehicle and islet transplant mice with IL - 33 treatment ( at day 7 , 30 and 80 post - islet transplantation ) . Data shown are the mean ± SEM ( n = 4 - 6 per group ) and a one - way ANOVA was performed ; * * * P < 0 . 001 .

O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-CELL_TYPE O O B-TISSUE O B-TISSUE O B-TISSUE B-EXP_ASSAY O O O B-TISSUE O B-ORGANISM O O O B-TISSUE O B-ORGANISM O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O 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-D) Proportion or numbers of CD4+Foxp3+ Tregs in the spleens, kidneys and islet grafts of normal, islet transplant mice receiving vehicle and islet transplant mice with IL-33 treatment (at day 7, 30 and 80 post-islet transplantation). Data shown are the mean ± SEM (n=4-6 per group) and a one-way ANOVA was performed; ***P<0.001.


( E ) Representative confocal microscopy images of immunostaining for CD4 , Foxp3 and insulin in islet grafts . Scale bar = 50 µm .

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(E) Representative confocal microscopy images of immunostaining for CD4, Foxp3 and insulin in islet grafts. Scale bar = 50 µm.


( F - H ) Proportion or numbers of ILC2s in the spleens , kidneys and islet graft of mice with or without IL - 33 treatment . Data shown are the mean ± SEM ( n = 4 - 6 per group ) and a one - way ANOVA was performed ; * * P < 0 . 01 , * * * P < 0 . 001 .

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(F-H) Proportion or numbers of ILC2s in the spleens, kidneys and islet graft of mice with or without IL-33 treatment. Data shown are the mean ± SEM (n=4-6 per group) and a one-way ANOVA was performed; **P<0.01, ***P<0.001.


( I ) Representative confocal microscopy images of immunostaining for CD127 , ST2 , CD3 and insulin in islet grafts . Scale bar = 50 µm .

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(I) Representative confocal microscopy images of immunostaining for CD127, ST2, CD3 and insulin in islet grafts. Scale bar = 50 µm.


( J ) The mRNA expression of IL - 25 , IL - 33 and TSLP in islet grafts of mice with or without IL - 33 treatment was examined by qPCR , and expressed relative to the control of each experiment . Data shown are the mean ± SEM ( n = 4 - 6 per group ) and a one - way ANOVA was performed ; * * P < 0 . 01 , * * * P < 0 . 001 .

O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-TISSUE B-EXP_ASSAY O B-ORGANISM O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(J) The mRNA expression of IL-25, IL-33 and TSLP in islet grafts of mice with or without IL-33 treatment was examined by qPCR, and expressed relative to the control of each experiment. Data shown are the mean ± SEM (n=4-6 per group) and a one-way ANOVA was performed; **P<0.01, ***P<0.001.


Proportion of CD4 + Foxp3 + Tregs ( B ) from the spleens and kidneys of islet transplant mice receiving vehicle , IL - 33 , IL - 33 / DT , IL - 33 / PC61 , or IL - 33 / DT / PC61 at day 5 post - islet transplantation . Data shown are the mean ± SEM ( n = 4 - 5 per group ) and a one - way ANOVA was performed ; NS : non significant , * * * P < 0 . 001 .

O O B-GENEPROD O B-GENEPROD O B-CELL_TYPE O O O O O B-TISSUE O B-TISSUE O B-TISSUE O B-ORGANISM O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Proportion of CD4+Foxp3+ Tregs (B) from the spleens and kidneys of islet transplant mice receiving vehicle, IL-33, IL-33/DT, IL-33/PC61, or IL-33/DT/PC61 at day 5 post-islet transplantation. Data shown are the mean ± SEM (n=4-5 per group) and a one-way ANOVA was performed; NS: non significant, ***P<0.001.


Proportion of Lin - GATA - 3 + ILC2s ( C ) from the spleens and kidneys of islet transplant mice receiving vehicle , IL - 33 , IL - 33 / DT , IL - 33 / PC61 , or IL - 33 / DT / PC61 at day 5 post - islet transplantation . Data shown are the mean ± SEM ( n = 4 - 5 per group ) and a one - way ANOVA was performed ; NS : non significant , * * * P < 0 . 001 .

O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-TISSUE O B-TISSUE O B-TISSUE O B-ORGANISM O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-SMALL_MOLECULE O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Proportion of Lin-GATA-3+ ILC2s (C) from the spleens and kidneys of islet transplant mice receiving vehicle, IL-33, IL-33/DT, IL-33/PC61, or IL-33/DT/PC61 at day 5 post-islet transplantation. Data shown are the mean ± SEM (n=4-5 per group) and a one-way ANOVA was performed; NS: non significant, ***P<0.001.


( D ) Islet graft survival of five groups of mice was assessed by monitoring blood glucose and calculated using the Kaplan - Meier method . Cumulative data from two independent experiments are shown . Statistical analysis was performed with a log - rank test . * P < 0 . 05 , * * P < 0 . 01 .

O O O B-TISSUE B-EXP_ASSAY I-EXP_ASSAY O O O O B-ORGANISM O O O O B-TISSUE B-SMALL_MOLECULE O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O 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) Islet graft survival of five groups of mice was assessed by monitoring blood glucose and calculated using the Kaplan-Meier method. Cumulative data from two independent experiments are shown. Statistical analysis was performed with a log-rank test. *P<0.05, **P<0.01.


( E ) Data are shown as blood glucose measurement in islet transplant mice treated with IL - 33 / DT , IL - 33 / PC61 , or IL - 33 / DT / PC61 . The horizontal black line indicates a BGL of 16 mmol / liter , the threshold for rejection . Each line represents one mouse .

O O O O O O O B-TISSUE B-SMALL_MOLECULE O O O O B-ORGANISM O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O B-ORGANISM O

(E) Data are shown as blood glucose measurement in islet transplant mice treated with IL-33/DT, IL-33/PC61, or IL-33/DT/PC61. The horizontal black line indicates a BGL of 16 mmol/liter, the threshold for rejection. Each line represents one mouse.


( A , B ) ILC210 and non - IL - 10 producing ILC2s were assessed in islet graft ( A ) and kidney ( B ) by intracellular IL - 10 staining at day 5 post islet transplantation . Data shown are the mean ± SEM ( n = 5 per group ) and an unpaired t - test was performed ; * * * P < 0 . 001 . ST2 : suppression of tumorigenicity 2 .

O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-TISSUE B-EXP_ASSAY O O O O B-TISSUE O O O O B-SUBCELLULAR B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O O O 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-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O

(A,B) ILC210 and non-IL-10 producing ILC2s were assessed in islet graft (A) and kidney (B) by intracellular IL-10 staining at day 5 post islet transplantation. Data shown are the mean ± SEM (n=5 per group) and an unpaired t-test was performed; ***P<0.001. ST2: suppression of tumorigenicity 2.


( C ) IL - 10 reporter C57BL / 6 mice were treated with PBS , IL - 33 alone or IL - 33 and IL - 2C daily for 5 consecutive days . Proportion of lin - CD127 + ST2 + IL - 10 - GFP ILC210 from the kidneys at day 3 after treatment in IL - 10 reporter C57BL / 6 mice . Data shown are the mean ± SEM ( n = 4 - 6 per group ) and a one - way ANOVA was performed ; * * * P < 0 . 001 .

O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-ORGANISM O O O B-SMALL_MOLECULE O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O B-TISSUE O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) IL-10 reporter C57BL/6 mice were treated with PBS, IL-33 alone or IL-33 and IL-2C daily for 5 consecutive days. Proportion of lin-CD127+ST2+IL-10-GFP ILC210 from the kidneys at day 3 after treatment in IL-10 reporter C57BL/6 mice. Data shown are the mean ± SEM (n=4-6 per group) and a one-way ANOVA was performed; ***P<0.001.


( D ) Kidney ILC2s isolated from normal IL - 10 reporter C57BL / 6 mice were cultured with Medium only or Medium with IL - 33 and IL - 2C for six days . Proportion of ILC210 was assessed by flow cytometry . Data shown are the mean ± SEM ( n = 6 per group ) and an unpaired t - test was performed ; * * * P < 0 . 001 . ST2 : suppression of tumorigenicity 2 .

O O O B-TISSUE O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-ORGANISM O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O 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-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O

(D) Kidney ILC2s isolated from normal IL-10 reporter C57BL/6 mice were cultured with Medium only or Medium with IL-33 and IL-2C for six days. Proportion of ILC210 was assessed by flow cytometry. Data shown are the mean ± SEM (n=6 per group) and an unpaired t-test was performed; ***P<0.001. ST2: suppression of tumorigenicity 2.


( E ) The secreted cytokine IL - 10 was analyzed via ELISA . Data shown are the mean ± SEM ( n = 6 per group ) and an unpaired t - test was performed ; * * * P < 0 . 001 .

O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) The secreted cytokine IL-10 was analyzed via ELISA. Data shown are the mean ± SEM (n=6 per group) and an unpaired t-test was performed; ***P<0.001.


( F ) Kidney ILC2s isolated from C57BL / 6 mice were cultured with Medium only or Medium with IL - 33 and IL - 2C for 30 minutes . The expression of phosphorylated STAT5 was examined in kidney ILC2s by flow cytometry . p - STAT5 ( red lines ) and isotype controls ( gray - filled areas ) are shown . Data represent the mean ± SEM of evaluations of MFI from each group ( n = 6 per group ) and an unpaired t - test was performed ; * * * P < 0 . 001 . MFI , mean fluorescence intensity .

O O O B-TISSUE O O O O O O B-ORGANISM O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O B-EXP_ASSAY B-GENEPROD O O O B-TISSUE O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O 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-EXP_ASSAY O O O O 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-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O

(F) Kidney ILC2s isolated from C57BL/6 mice were cultured with Medium only or Medium with IL-33 and IL-2C for 30 minutes. The expression of phosphorylated STAT5 was examined in kidney ILC2s by flow cytometry. p-STAT5 (red lines) and isotype controls (gray-filled areas) are shown. Data represent the mean ± SEM of evaluations of MFI from each group (n=6 per group) and an unpaired t-test was performed; ***P<0.001. MFI, mean fluorescence intensity.


( G ) The kidney ILC2s were cultured with Medium , IL - 33 / IL - 2C , or IL - 33 / IL - 2C and STAT5 inhibitor ( STAT5 - IN ) for three days . The secreted cytokine IL - 10 was analyzed via ELISA . Data shown are the mean ± SEM ( n = 6 per group ) and a one - way ANOVA was performed ; * * * P < 0 . 001 .

O O O O B-TISSUE O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G) The kidney ILC2s were cultured with Medium, IL-33/IL-2C, or IL-33/IL-2C and STAT5 inhibitor (STAT5-IN) for three days. The secreted cytokine IL-10 was analyzed via ELISA. Data shown are the mean ± SEM (n=6 per group) and a one-way ANOVA was performed; ***P<0.001.


( A ) ILC210 and non - ILC210 were isolated from ex vivo - expanded kidney ILC2 by flow sorting . ILC210 were transfected with control ( ILC210 - C ) or IL - 10 CRISPR - Cas9 ( ILC210 - IL - 10 ) . IL - 10 was measured in culture supernatant of ILC210 - C and ILC210 - IL - 10 via ELISA . Data shown are the mean ± SEM ( n = 6 per group ) and an unpaired t - test was performed ; * * * P < 0 . 001 .

O O O O O O O O O O O O O O O B-TISSUE O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-EXP_ASSAY O O O O O O O O O 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) ILC210 and non-ILC210 were isolated from ex vivo-expanded kidney ILC2 by flow sorting. ILC210 were transfected with control (ILC210-C) or IL-10 CRISPR-Cas9 (ILC210-IL-10). IL-10 was measured in culture supernatant of ILC210-C and ILC210-IL-10 via ELISA. Data shown are the mean ± SEM (n=6 per group) and an unpaired t-test was performed; ***P<0.001.


( C ) Transfused ILC2s ( CFSE labeled , Green ) were observed in islet graft at day 5 post islet transplantation . Scale bar = 50 µm . The numbers of CFSE - labeled ILC2s in the islet graft were counted . Data shown are the mean ± SEM and a one - way ANOVA was performed ( n = 4 per group ) . NS , not significant .

O O O O O O B-SMALL_MOLECULE O O O O O O O B-TISSUE B-EXP_ASSAY O O O O O O O O O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O B-TISSUE B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Transfused ILC2s (CFSE labeled, Green) were observed in islet graft at day 5 post islet transplantation. Scale bar = 50 µm. The numbers of CFSE-labeled ILC2s in the islet graft were counted. Data shown are the mean ± SEM and a one-way ANOVA was performed (n=4 per group). NS, not significant.


( D ) The mRNA expression of IL - 10 in islet grafts at day 5 post islet transplantation was examined by qPCR . Data shown are the mean ± SEM ( n = 4 per group ) and a one - way ANOVA was performed ; * * P < 0 . 01 , * * * P < 0 . 001 .

O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-TISSUE B-EXP_ASSAY O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O 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) The mRNA expression of IL-10 in islet grafts at day 5 post islet transplantation was examined by qPCR. Data shown are the mean ± SEM (n=4 per group) and a one-way ANOVA was performed; **P<0.01, ***P<0.001.


( E ) Islet graft survival of four groups of mice was assessed by monitoring blood glucose and calculated using the Kaplan - Meier method . Cumulative data from two independent experiments are shown . Statistical analysis was performed with a log - rank test . * * * P < 0 . 001 .

O O O B-TISSUE B-EXP_ASSAY I-EXP_ASSAY O O O O B-ORGANISM O O O O B-TISSUE B-SMALL_MOLECULE O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) Islet graft survival of four groups of mice was assessed by monitoring blood glucose and calculated using the Kaplan-Meier method. Cumulative data from two independent experiments are shown. Statistical analysis was performed with a log-rank test. ***P<0.001.


( F ) CD4 + T cells isolated from C57BL / 6 splenocytes were cultured with irradiated splenocytes derived from BALB / c in the presence of ILC210 at the indicated ratios for 4 days . CD4 + T cells proliferation was assessed using [ 3H ] - thymidine incorporation assays . Data shown are the mean ± SEM ( n = 4 - 6 per group ) .

O O O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O O O O B-CELL_TYPE O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE B-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O

(F) CD4+ T cells isolated from C57BL/6 splenocytes were cultured with irradiated splenocytes derived from BALB/c in the presence of ILC210 at the indicated ratios for 4 days. CD4+ T cells proliferation was assessed using [3H]-thymidine incorporation assays. Data shown are the mean ± SEM (n=4-6 per group).


( G ) Neutralizing anti - IL - 10 antibodies or genetic ablation of IL - 10 were used to block the effect of ILC210 on CD4 + T cells proliferation . Data shown are the mean ± SEM ( n = 6 per group ) and an unpaired t - test was performed . * * * P < 0 . 001 .

O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G) Neutralizing anti-IL-10 antibodies or genetic ablation of IL-10 were used to block the effect of ILC210 on CD4+ T cells proliferation. Data shown are the mean ± SEM (n=6 per group) and an unpaired t-test was performed. ***P<0.001.


( B ) Islet graft survival of five groups of mice was assessed by monitoring blood glucose and calculated using the Kaplan - Meier method . Cumulative data from two independent experiments are shown . Statistical analysis was performed with a log - rank test . * * * P < 0 . 001 .

(B) Islet graft survival of five groups of mice was assessed by monitoring blood glucose and calculated using the Kaplan-Meier method. Cumulative data from two independent experiments are shown. Statistical analysis was performed with a log-rank test. ***P<0.001.


( C ) To track ILC210 in vivo , CD45 . 2 + ILC210 were co - transplanted with islets in diabetic CD45 . 1 + C57BL / 6 mice . Representative FACS analysis showing the proportion of locally transplanted ILC210 ( CD45 . 2 + ST2 + ) in the total CD45 + cell compartment from islet graft at day 5 and 80 post - islet transplantation or at the day when grafts were considered rejected .

O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-TISSUE O B-DISEASE B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-ORGANISM O O B-EXP_ASSAY O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O O O B-TISSUE B-EXP_ASSAY O O O O O O O O O O O O O O B-EXP_ASSAY O O O O

(C) To track ILC210 in vivo, CD45.2+ ILC210 were co-transplanted with islets in diabetic CD45.1+ C57BL/6 mice. Representative FACS analysis showing the proportion of locally transplanted ILC210 (CD45.2+ST2+) in the total CD45+ cell compartment from islet graft at day 5 and 80 post-islet transplantation or at the day when grafts were considered rejected.


( D , E ) Proportion and numbers of locally transplanted CD45 . 2 + ST2 + ILC210 in islet grafts of islet transplant mice over time . Data shown are the mean ± SEM ( n = 4 - 5 per group ) and a one - way ANOVA was performed , * * P < 0 . 01 , * * * P < 0 . 001 .

O O O O O O O B-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O B-TISSUE B-EXP_ASSAY O B-TISSUE O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O 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,E) Proportion and numbers of locally transplanted CD45.2+ST2+ ILC210 in islet grafts of islet transplant mice over time. Data shown are the mean ± SEM (n=4-5 per group) and a one-way ANOVA was performed, **P<0.01, ***P<0.001.


( F ) The expression of IL - 10 - GFP was examined in locally transplanted CD45 . 2 + ST2 + ILC210 by flow cytometry . IL - 10 - GFP ( red lines ) and controls ( gray - filled areas ) are shown . Data represent the mean ± SEM of evaluations of MFI from each group ( n = 4 - 5 per group ) and a one - way ANOVA was performed ; * * * P < 0 . 001 . MFI , mean fluorescence intensity .

O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD 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-EXP_ASSAY O O O O O O 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-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O

(F) The expression of IL-10-GFP was examined in locally transplanted CD45.2+ST2+ ILC210 by flow cytometry. IL-10-GFP (red lines) and controls (gray-filled areas) are shown. Data represent the mean ± SEM of evaluations of MFI from each group (n=4-5 per group) and a one-way ANOVA was performed; ***P<0.001. MFI, mean fluorescence intensity.


B . ST2 cells were stimulated for 15 minutes with SF - IL - 17 ( 500 ng / ml ) , solubilized and IL - 17RSC was isolated via consecutive Flag and Strep immunoprecipitation and analyzed by MS . As a control , cells were first solubilized and SF - IL - 17 was added only post lysis . Number of identified peptides ( unique + razor ) and iBAQ intensities for each protein in five independent experiments are shown .

O O B-CELL_LINE O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O O O B-EXP_ASSAY O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O 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. ST2 cells were stimulated for 15 minutes with SF-IL-17 (500 ng/ml), solubilized and IL-17RSC was isolated via consecutive Flag and Strep immunoprecipitation and analyzed by MS. As a control, cells were first solubilized and SF-IL-17 was added only post lysis. Number of identified peptides (unique + razor) and iBAQ intensities for each protein in five independent experiments are shown.


C . The stoichiometry of IL - 17RSC calculated as the ratio between iBAQ intensities of individual IL - 17RSC components to iBAQ intensity of IL - 17RC . The recruitment of kinases TBK1 and IKKε as compared to related kinases IKKα and IKKβ is significantly enhanced . Mean from five performed MS experiments is shown , the statistical significance was determined using unpaired two ‐ tailed Student ' s t ‐ test .

O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O

C. The stoichiometry of IL-17RSC calculated as the ratio between iBAQ intensities of individual IL-17RSC components to iBAQ intensity of IL-17RC. The recruitment of kinases TBK1 and IKKε as compared to related kinases IKKα and IKKβ is significantly enhanced. Mean from five performed MS experiments is shown, the statistical significance was determined using unpaired two‐tailed Student's t‐test.


D . ST2 cells were stimulated with IL - 17 ( 500 ng / ml ) , TNF ( 50 ng / ml ) , or IL - 1α ( 50 ng / ml ) for indicated time points and activation of signaling pathways was analyzed by immunoblotting . A representative of two independent experiments is shown .

O O B-CELL_LINE O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O

D. ST2 cells were stimulated with IL-17 (500 ng/ml), TNF (50 ng/ml), or IL-1α (50 ng/ml) for indicated time points and activation of signaling pathways was analyzed by immunoblotting. A representative of two independent experiments is shown.


ST2 cells were either left untreated ( A ) followed by stimulation with IL - 17 ( 500 ng / ml ) for 2 hours . Transcription response induced by IL - 17 stimulation as compared to unstimulated cells was analyzed by RNA sequencing . In red are transcripts considered to be significantly changed ( log2 fold change > 1 or < - 1 , - log10 Benjamini - Hochberg adjusted p - value > 2 , based on the analysis of three independent experiments ; tringle is used for transcripts with - log10 adjusted p - value > 100 ) . Names of several significantly upregulated transcripts are indicated .

B-CELL_LINE O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

ST2 cells were either left untreated (A) followed by stimulation with IL-17 (500 ng/ml) for 2 hours. Transcription response induced by IL-17 stimulation as compared to unstimulated cells was analyzed by RNA sequencing. In red are transcripts considered to be significantly changed (log2 fold change > 1 or < -1, - log10 Benjamini-Hochberg adjusted p-value > 2, based on the analysis of three independent experiments; tringle is used for transcripts with - log10 adjusted p-value > 100). Names of several significantly upregulated transcripts are indicated.


ST2 cells were pretreated with TBK1 / IKKε inhibitor MRT67307 ( 2 µM ) for 30 minutes ( B ) , followed by stimulation with IL - 17 ( 500 ng / ml ) for 2 hours . Transcription response induced by IL - 17 stimulation as compared to unstimulated cells was analyzed by RNA sequencing . In red are transcripts considered to be significantly changed ( log2 fold change > 1 or < - 1 , - log10 Benjamini - Hochberg adjusted p - value > 2 , based on the analysis of three independent experiments ; tringle is used for transcripts with - log10 adjusted p - value > 100 ) . Names of several significantly upregulated transcripts are indicated .

B-CELL_LINE O O O O B-GENEPROD O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

ST2 cells were pretreated with TBK1/IKKε inhibitor MRT67307 (2 µM) for 30 minutes (B), followed by stimulation with IL-17 (500 ng/ml) for 2 hours. Transcription response induced by IL-17 stimulation as compared to unstimulated cells was analyzed by RNA sequencing. In red are transcripts considered to be significantly changed (log2 fold change > 1 or < -1, - log10 Benjamini-Hochberg adjusted p-value > 2, based on the analysis of three independent experiments; tringle is used for transcripts with - log10 adjusted p-value > 100). Names of several significantly upregulated transcripts are indicated.


D - E . Comparison of IL - 17 - induced transcriptional response in presence or absence of TBK1 / IKKε inhibitor . ( D ) Transcripts that are significantly changed in both conditions . ( E ) Transcripts that pass significance threshold for induction only in presence of TBK1 / IKKε inhibitor . Dashed lines indicate significantly upregulated transcripts ( log2 fold change > 1 ) ; red lines separates transcripts that are more upregulated upon IL - 17 stimulation in the presence of TBK1 / IKKε inhibitor as compared to IL - 17 alone .

O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O 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-GENEPROD O B-GENEPROD O 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-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O

D-E. Comparison of IL-17-induced transcriptional response in presence or absence of TBK1/IKKε inhibitor. (D) Transcripts that are significantly changed in both conditions. (E) Transcripts that pass significance threshold for induction only in presence of TBK1/IKKε inhibitor. Dashed lines indicate significantly upregulated transcripts (log2 fold change > 1); red lines separates transcripts that are more upregulated upon IL-17 stimulation in the presence of TBK1/IKKε inhibitor as compared to IL-17 alone.


F . ST2 cells pretreated or not with TBK1 / IKKε inhibitor MRT67307 ( 2 µM ) were left unstimulated or stimulated with IL - 17 ( 500 ng / ml ) for 2 hours and induction of mRNA for selected genes was analyzed by real time PCR . Mean + SEM from five independent experiments is shown , statistical significance was determined using unpaired two ‐ tailed Student ' s t ‐ test .

O O B-CELL_LINE O O O O O B-GENEPROD O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O

F. ST2 cells pretreated or not with TBK1/IKKε inhibitor MRT67307 (2 µM) were left unstimulated or stimulated with IL-17 (500 ng/ml) for 2 hours and induction of mRNA for selected genes was analyzed by real time PCR. Mean + SEM from five independent experiments is shown, statistical significance was determined using unpaired two‐tailed Student's t‐test.


G . ST2 cells were pretreated or not with TBK1 / IKKε inhibitor MRT67307 ( 2 µM ) , stimulated with IL17 ( 500 ng / ml ) for indicated time points and analyzed by immunoblotting .

O O B-CELL_LINE O O O O O O B-GENEPROD O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O O O O O O O O O O O O O B-EXP_ASSAY O

G. ST2 cells were pretreated or not with TBK1/IKKε inhibitor MRT67307 (2 µM), stimulated with IL17 (500 ng/ml) for indicated time points and analyzed by immunoblotting.


H . ST2 wild type , TBK1 KO , IKKε KO , or cells lacking both kinases ( DKO ) were stimulated with IL - 17 ( 500 ng / ml ) as indicated and analyzed by immunoblotting .

O O B-CELL_LINE O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O B-EXP_ASSAY O

H. ST2 wild type, TBK1 KO, IKKε KO, or cells lacking both kinases (DKO) were stimulated with IL-17 (500 ng/ml) as indicated and analyzed by immunoblotting.


I . TBK1 / IKKε DKO cells reconstituted with either wild type or kinase dead mutant ( K38A ) version of both kinases were stimulated with IL - 17 ( 500 ng / ml ) as indicated and analyzed by immunoblotting .

O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O B-EXP_ASSAY O

I. TBK1/IKKε DKO cells reconstituted with either wild type or kinase dead mutant (K38A) version of both kinases were stimulated with IL-17 (500 ng/ml) as indicated and analyzed by immunoblotting.


ST2 wild type or TBK1 / IKKε DKO cells were stimulated for 15 minutes with SF - IL - 17 ( 500 ng / ml ) , solubilized and IL - 17RSC was isolated via consecutive Flag and Strep immunoprecipitation and analyzed by MS . ( A ) The heatmap shows the row - normalized Z - Score calculated from log2 transformed iBAQ intensities from five independent experiments .

B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O

ST2 wild type or TBK1/IKKε DKO cells were stimulated for 15 minutes with SF-IL-17 (500 ng/ml), solubilized and IL-17RSC was isolated via consecutive Flag and Strep immunoprecipitation and analyzed by MS. (A) The heatmap shows the row-normalized Z-Score calculated from log2 transformed iBAQ intensities from five independent experiments.


ST2 wild type or TBK1 / IKKε DKO cells were stimulated for 15 minutes with SF - IL - 17 ( 500 ng / ml ) , solubilized and IL - 17RSC was isolated via consecutive Flag and Strep immunoprecipitation and analyzed by MS . ( B ) The ratio between iBAQ intensities of selected IL - 17RSC components to iBAQ intensity of IL - 17RC . Mean + SEM from five independent experiments is shown , statistical significance was determined by two ‐ tailed Mann - Whitney test .

B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O O O B-EXP_ASSAY O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O

ST2 wild type or TBK1/IKKε DKO cells were stimulated for 15 minutes with SF-IL-17 (500 ng/ml), solubilized and IL-17RSC was isolated via consecutive Flag and Strep immunoprecipitation and analyzed by MS. (B) The ratio between iBAQ intensities of selected IL-17RSC components to iBAQ intensity of IL-17RC. Mean + SEM from five independent experiments is shown, statistical significance was determined by two‐tailed Mann-Whitney test.


C . ST2 wild type or TBK1 / IKKε DKO cells were stimulated with SF - IL - 17 ( 500 ng / ml ) for 15 minutes or were left unstimulated and IL - 17 was added post lysis . Lysates were subjected to anti - Flag immunoprecipitation to isolate IL - 17RSC and samples were analyzed by immunoblotting . ACT1 phosphorylation detected as upper band in WT cells is absent in TBK1 / IKKε DKO cells .

O O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-EXP_ASSAY O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O

C. ST2 wild type or TBK1/IKKε DKO cells were stimulated with SF-IL-17 (500 ng/ml) for 15 minutes or were left unstimulated and IL-17 was added post lysis. Lysates were subjected to anti-Flag immunoprecipitation to isolate IL-17RSC and samples were analyzed by immunoblotting. ACT1 phosphorylation detected as upper band in WT cells is absent in TBK1/IKKε DKO cells.


D . Cells were pretreated with TAK1 inhibitor 7 - oxozeanol ( 2 µM ) and / or TBK1 / IKKε inhibitor MRT67307 ( 2 µM ) , stimulated with IL - 17 ( 500 ng / ml ) for indicated time points and lysates were analyzed by immunoblotting .

O O O O O O B-GENEPROD O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-GENEPROD O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O B-EXP_ASSAY O

D. Cells were pretreated with TAK1 inhibitor 7-oxozeanol (2 µM) and/or TBK1/IKKε inhibitor MRT67307 (2 µM), stimulated with IL-17 (500 ng/ml) for indicated time points and lysates were analyzed by immunoblotting.


E . ST2 wild type or HOIP - deficient cells were pretreated with TBK1 / IKKε inhibitor MRT67307 ( 2 µM ) , stimulated with IL - 17 ( 500 ng / ml ) as indicated , solubilized , and analyzed by immunoblotting . * indicates unspecific band .

O O B-CELL_LINE O O O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O

E. ST2 wild type or HOIP-deficient cells were pretreated with TBK1/IKKε inhibitor MRT67307 (2 µM), stimulated with IL-17 (500 ng/ml) as indicated, solubilized, and analyzed by immunoblotting. * indicates unspecific band.


F . ST2 cells pretreated or not with TBK1 / IKKε inhibitor MRT67307 were stimulated with SF - IL - 17 for 15 minutes or were left unstimulated and IL - 17 was added post lysis . IL - 17RSC was isolated and analyzed via immunoblotting .

O O B-CELL_LINE O O O O O B-GENEPROD O B-GENEPROD O B-SMALL_MOLECULE O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-EXP_ASSAY O

F. ST2 cells pretreated or not with TBK1/IKKε inhibitor MRT67307 were stimulated with SF-IL-17 for 15 minutes or were left unstimulated and IL-17 was added post lysis. IL-17RSC was isolated and analyzed via immunoblotting.


A . ST2 wild type and ACT1 - deficient cells were stimulated for 15 minutes with SF - IL - 17 ( 500 ng / ml ) , solubilized and IL - 17RSC was isolated via consecutive Flag and Strep immunoprecipitation and analyzed by MS . As a control , cells were first solubilized and SF - IL - 17 was added only post lysis . Number of identified peptides ( unique + razor ) and iBAQ intensities for each protein in two independent experiments are shown .

O O B-CELL_LINE O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O O O B-EXP_ASSAY O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O 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. ST2 wild type and ACT1-deficient cells were stimulated for 15 minutes with SF-IL-17 (500 ng/ml), solubilized and IL-17RSC was isolated via consecutive Flag and Strep immunoprecipitation and analyzed by MS. As a control, cells were first solubilized and SF-IL-17 was added only post lysis. Number of identified peptides (unique + razor) and iBAQ intensities for each protein in two independent experiments are shown.


B . ST2 wild type , ACT1 KO or TRAF6 KO cells were stimulated with SF - IL - 17 ( 500 ng / ml ) as indicated or were left unstimulated and IL - 17 was added post lysis . Lysates were subjected to anti - Flag immunoprecipitation to isolate IL - 17RSC and samples were analyzed by immunoblotting .

O O B-CELL_LINE O O O B-GENEPROD O O B-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-EXP_ASSAY O

B. ST2 wild type, ACT1 KO or TRAF6 KO cells were stimulated with SF-IL-17 (500 ng/ml) as indicated or were left unstimulated and IL-17 was added post lysis. Lysates were subjected to anti-Flag immunoprecipitation to isolate IL-17RSC and samples were analyzed by immunoblotting.


C . ST2 cells deficient in TRAF6 reconstituted with TRAF6 ( WT ) or enzymatically inactive TRAF6 ( C70A ) were stimulated for 15 minutes with SF - IL - 17 ( 500 ng / ml ) , solubilized and IL - 17RSC was isolated via consecutive Flag and Strep immunoprecipitation and analyzed by MS . Number of identified peptides ( unique + razor ) and iBAQ intensities for each protein in two independent experiments are shown .

O O B-CELL_LINE O O O B-GENEPROD O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O

C. ST2 cells deficient in TRAF6 reconstituted with TRAF6(WT) or enzymatically inactive TRAF6(C70A) were stimulated for 15 minutes with SF-IL-17 (500 ng/ml), solubilized and IL-17RSC was isolated via consecutive Flag and Strep immunoprecipitation and analyzed by MS. Number of identified peptides (unique + razor) and iBAQ intensities for each protein in two independent experiments are shown.


D . TRAF6 deficient cells reconstituted with TRAF6 ( WT ) , TRAF6 ( C70A ) or empty vector were stimulated with SF - IL - 17 ( 500 ng / ml ) as indicated or were left unstimulated and IL - 17 was added post lysis . IL - 17RSC was isolated from lysates by anti - Flag immunoprecipitation and analyzed by immunoblotting . Short and long exposure of the same membrane stained for Act1 are shown for lysate and IP samples , respectively .

O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD B-EXP_ASSAY O O O B-EXP_ASSAY O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O B-EXP_ASSAY O O O O

D. TRAF6 deficient cells reconstituted with TRAF6(WT), TRAF6(C70A) or empty vector were stimulated with SF-IL-17 (500 ng/ml) as indicated or were left unstimulated and IL-17 was added post lysis. IL-17RSC was isolated from lysates by anti-Flag immunoprecipitation and analyzed by immunoblotting. Short and long exposure of the same membrane stained for Act1 are shown for lysate and IP samples, respectively.


TRAF6 deficient cells reconstituted with the indicated constructs were pretreated or not with TBK1 / IKKε inhibitor MRT67307 ( 2 µM ) and stimulated with IL - 17 ( 500 ng / ml ) for indicated time . ( E ) The activation of signaling pathways analyzed by immunoblotting .

B-GENEPROD O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O

TRAF6 deficient cells reconstituted with the indicated constructs were pretreated or not with TBK1/IKKε inhibitor MRT67307 (2 µM) and stimulated with IL-17 (500 ng/ml) for indicated time. (E) The activation of signaling pathways analyzed by immunoblotting.


TRAF6 deficient cells reconstituted with the indicated constructs were pretreated or not with TBK1 / IKKε inhibitor MRT67307 ( 2 µM ) and stimulated with IL - 17 ( 500 ng / ml ) for indicated time . The induction of mRNA for selected genes analyzed by real time PCR . Mean + SEM from four independent experiments is shown , statistical significance was determined using unpaired two ‐ tailed Student ' s t ‐ test .

B-GENEPROD O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O

TRAF6 deficient cells reconstituted with the indicated constructs were pretreated or not with TBK1/IKKε inhibitor MRT67307 (2 µM) and stimulated with IL-17 (500 ng/ml) for indicated time. The induction of mRNA for selected genes analyzed by real time PCR. Mean + SEM from four independent experiments is shown, statistical significance was determined using unpaired two‐tailed Student's t‐test.


G . ST2 cells wild type or NEMO KO were stimulated with IL - 17 ( 500 ng / ml ) for indicated time points , solubilized and analyzed by immunoblotting .

O O B-CELL_LINE O O O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O B-EXP_ASSAY O

G. ST2 cells wild type or NEMO KO were stimulated with IL-17 (500 ng/ml) for indicated time points, solubilized and analyzed by immunoblotting.


ST2 wild type or NEMO KO cells were stimulated with SF - IL - 17 ( 500 ng / ml ) as indicated or were left unstimulated and IL - 17 was added post lysis . Lysates were subjected to anti - Flag immunoprecipitation to isolate IL - 17RSC . Samples were analyzed by immunoblotting ( H ) .

B-CELL_LINE O O O B-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-EXP_ASSAY O O O O

ST2 wild type or NEMO KO cells were stimulated with SF-IL-17 (500 ng/ml) as indicated or were left unstimulated and IL-17 was added post lysis. Lysates were subjected to anti-Flag immunoprecipitation to isolate IL-17RSC. Samples were analyzed by immunoblotting (H).


ST2 wild type or NEMO KO cells were stimulated with SF - IL - 17 or were left unstimulated and IL - 17 was added post lysis . results from four independent experiments were quantified by densitometry and mean + SEM from four independent experiments is shown ( I ) .

B-CELL_LINE O O O B-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O

ST2 wild type or NEMO KO cells were stimulated with SF-IL-17 or were left unstimulated and IL-17 was added post lysis. results from four independent experiments were quantified by densitometry and mean + SEM from four independent experiments is shown (I).


ST2 wild type or NEMO KO cells were stimulated for 15 minutes with SF - IL - 17 ( 500 ng / ml ) , solubilized and IL - 17RSC was isolated via consecutive Flag and Strep immunoprecipitation and analyzed by MS . ( A ) The heatmap shows the row - normalized Z - Score calculated from log2 transformed iBAQ values from two independent experiments .

B-CELL_LINE O O O B-GENEPROD O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O

ST2 wild type or NEMO KO cells were stimulated for 15 minutes with SF-IL-17 (500 ng/ml), solubilized and IL-17RSC was isolated via consecutive Flag and Strep immunoprecipitation and analyzed by MS. (A) The heatmap shows the row-normalized Z-Score calculated from log2 transformed iBAQ values from two independent experiments.


ST2 wild type or NEMO KO cells were stimulated for 15 minutes with SF - IL - 17 ( 500 ng / ml ) , solubilized and IL - 17RSC was isolated via consecutive Flag and Strep immunoprecipitation and analyzed by MS . ( B ) The ratio between iBAQ intensities of selected IL - 17RSC components to iBAQ intensity of IL - 17RC . Mean + SEM from two independent experiments is shown .

B-CELL_LINE O O O B-GENEPROD O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O O O B-EXP_ASSAY O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O

ST2 wild type or NEMO KO cells were stimulated for 15 minutes with SF-IL-17 (500 ng/ml), solubilized and IL-17RSC was isolated via consecutive Flag and Strep immunoprecipitation and analyzed by MS. (B) The ratio between iBAQ intensities of selected IL-17RSC components to iBAQ intensity of IL-17RC. Mean + SEM from two independent experiments is shown.


ST2 wild type or NEMO KO cells were stimulated with SF - IL - 17 ( 500 ng / ml ) as indicated or were left unstimulated and IL - 17 was added post lysis . Lysates were subjected to anti - Flag immunoprecipitation to isolate IL - 17RSC ( C ) and analyzed by immunoblotting .

B-CELL_LINE O O O B-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-EXP_ASSAY O

ST2 wild type or NEMO KO cells were stimulated with SF-IL-17 (500 ng/ml) as indicated or were left unstimulated and IL-17 was added post lysis. Lysates were subjected to anti-Flag immunoprecipitation to isolate IL-17RSC (C) and analyzed by immunoblotting.


ST2 wild type or NEMO KO cells were stimulated with SF - IL - 17 ( 500 ng / ml ) as indicated or were left unstimulated and IL - 17 was added post lysis . Lysates were tested for activation of signaling pathways ( D ) and analyzed by immunoblotting .

B-CELL_LINE O O O B-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O

ST2 wild type or NEMO KO cells were stimulated with SF-IL-17 (500 ng/ml) as indicated or were left unstimulated and IL-17 was added post lysis. Lysates were tested for activation of signaling pathways (D) and analyzed by immunoblotting.


E . ST2 cells were pretreated with TBK1 / IKKε inhibitor MRT67307 ( 2 µM ) or IKKα / IKKβ inhibitor TPCA1 ( 10 µM ) , stimulated with IL - 17 ( 500 ng / ml ) as indicated and analyzed by immunoblotting .

O O B-CELL_LINE O O O O B-GENEPROD O B-GENEPROD O B-SMALL_MOLECULE O O O O O B-GENEPROD O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O B-EXP_ASSAY O

E. ST2 cells were pretreated with TBK1/IKKε inhibitor MRT67307 (2 µM) or IKKα/IKKβ inhibitor TPCA1 (10 µM), stimulated with IL-17 (500 ng/ml) as indicated and analyzed by immunoblotting.


F . ST2 wild type or NEMO KO cells were stimulated with TNF ( 50 ng / ml ) for indicated time points and lysates were analyzed by immunoblotting .

O O B-CELL_LINE O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-EXP_ASSAY O

F. ST2 wild type or NEMO KO cells were stimulated with TNF (50 ng/ml) for indicated time points and lysates were analyzed by immunoblotting.


B . Analysis of ACT1 structure using three different algorithms predicting disordered regions . Detected phosphorylation sites are all in the disordered region of the protein .

O O O O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O

B. Analysis of ACT1 structure using three different algorithms predicting disordered regions. Detected phosphorylation sites are all in the disordered region of the protein.


ACT1 KO cells were reconstituted with either wild type ACT1 or indicated mutants or ACT1 with all nine identified phospho - sites mutated to alanines ( 9ST mut ) . Cells were stimulated with IL - 17 ( 500 ng / ml ) as indicated and lysates were analyzed by immunoblotting . * indicates nonspecific band .

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ACT1 KO cells were reconstituted with either wild type ACT1 or indicated mutants or ACT1 with all nine identified phospho-sites mutated to alanines (9ST mut). Cells were stimulated with IL-17 (500 ng/ml) as indicated and lysates were analyzed by immunoblotting. * indicates nonspecific band.


E . ACT1 KO cells expressing either ACT1 ( WT ) or ACT1 ( 9ST mut ) were stimulated with SF - IL - 17 ( 500 ng / ml ) as indicated or were left unstimulated and IL - 17 was added post lysis . IL - 17RSC was isolated via anti - Flag immunoprecipitation and analyzed by immunoblotting .

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E. ACT1 KO cells expressing either ACT1(WT) or ACT1(9ST mut) were stimulated with SF-IL-17 (500 ng/ml) as indicated or were left unstimulated and IL-17 was added post lysis. IL-17RSC was isolated via anti-Flag immunoprecipitation and analyzed by immunoblotting.


F . ACT1 KO cells were reconstituted with either ACT1 ( WT ) or ACT1 ( Δ20 - 380 ) . Cells were stimulated with IL - 17 ( 500 ng / ml ) as indicated and lysates were analyzed by immunoblotting . * indicates nonspecific band .

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F. ACT1 KO cells were reconstituted with either ACT1(WT) or ACT1(Δ20-380). Cells were stimulated with IL-17 (500 ng/ml) as indicated and lysates were analyzed by immunoblotting. * indicates nonspecific band.


G . Analysis of iBAQ intensity of ACT1 normalized to iBAQ intensity of IL - 17RC . Data are composite of all 14 MS analyses of IL - 17RSC from wild type ST2 cells performed in this study . Mean + SEM is shown .

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G. Analysis of iBAQ intensity of ACT1 normalized to iBAQ intensity of IL-17RC. Data are composite of all 14 MS analyses of IL-17RSC from wild type ST2 cells performed in this study. Mean + SEM is shown.


( B ) ACE2 gene expression summary in human tissues in 61 different tissues and cells in NX . Cut - off for what is regarded as expressed was set to 1 . 0 NX .

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(B) ACE2 gene expression summary in human tissues in 61 different tissues and cells in NX. Cut-off for what is regarded as expressed was set to 1.0 NX.


Representative images of 20 tissue types and histological structures stained on consecutive sections with immunohistochemistry using two antibodies targeting human ACE2 protein ( brown ) , and counterstained with hematoxylin ( blue ) . Most intense antibody staining was observed in microvilli of the intestinal tract and renal proximal tubules , in membranes of gallbladder epithelium , epididymis epithelium , testicular Sertoli cells and Leydig cells , a subset of glandular cells in seminal vesicle and cytoplasm of cardiomyocytes , with HPA000288 also staining the cardiac muscle fibers , while MAB933 only showed staining in a few cells . Distinct ACE2 staining for both antibodies was also present in cornea and conjunctiva of the eye , interlobular pancreatic ducts , as well as in placental villi , both in cytotrophoblasts , syncytiotrophoblasts , and also in extravillous trophoblasts , while placenta decidua was negative . ACE2 staining could be observed at the base of ciliated fallopian tube epithelium , however only for one of the antibodies . Note that ACE2 protein expression was less prominent in the crypts of the mucosal intestinal layer . ACE2 also stained positive in endothelial cells and pericytes in several tissues , see Fallopian , thyroid , parathyroid , adrenal gland , pancreas and heart . Scale bar = 50µm . Scale bar in dashed squares = 10 µm . ( Brunner = Brunner ' s gland , EVT = extravillous trophoblasts , endo = endothelial cells ) .

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Representative images of 20 tissue types and histological structures stained on consecutive sections with immunohistochemistry using two antibodies targeting human ACE2 protein (brown), and counterstained with hematoxylin (blue). Most intense antibody staining was observed in microvilli of the intestinal tract and renal proximal tubules, in membranes of gallbladder epithelium, epididymis epithelium, testicular Sertoli cells and Leydig cells, a subset of glandular cells in seminal vesicle and cytoplasm of cardiomyocytes, with HPA000288 also staining the cardiac muscle fibers, while MAB933 only showed staining in a few cells. Distinct ACE2 staining for both antibodies was also present in cornea and conjunctiva of the eye, interlobular pancreatic ducts, as well as in placental villi, both in cytotrophoblasts, syncytiotrophoblasts, and also in extravillous trophoblasts, while placenta decidua was negative. ACE2 staining could be observed at the base of ciliated fallopian tube epithelium, however only for one of the antibodies. Note that ACE2 protein expression was less prominent in the crypts of the mucosal intestinal layer. ACE2 also stained positive in endothelial cells and pericytes in several tissues, see Fallopian, thyroid, parathyroid, adrenal gland, pancreas and heart. Scale bar = 50µm. Scale bar in dashed squares = 10 µm. (Brunner = Brunner's gland, EVT = extravillous trophoblasts, endo=endothelial cells).


Representative images of human respiratory tissues , all stained on consecutive sections , with immunohistochemistry using two antibodies targeting human ACE2 protein ( brown ) , and counterstained with hematoxylin ( blue ) . Respiratory tissues were composed of different structures in nasal mucosa , bronchus , smaller bronchioles and lung tissue . ACE2 staining could be observed at the base of ciliated cells in both nasal mucosa and bronchial epithelium ( arrowheads ) . Rare ACE2 staining was present in a few alveolar cells ( arrows ) . No staining was observed in nasal squamous epithelium , bronchioles , or submucosal glands in either tissue . Gender and age are shown for all individuals . Red and green colored squares mark the positions in the TMA cores shown as magnifications . Scale bar = 50µm . Scale bar for images in dashed squares = 10 µm . ( re = respiratory ; sq = squamous ) .

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Representative images of human respiratory tissues, all stained on consecutive sections, with immunohistochemistry using two antibodies targeting human ACE2 protein (brown), and counterstained with hematoxylin (blue). Respiratory tissues were composed of different structures in nasal mucosa, bronchus, smaller bronchioles and lung tissue. ACE2 staining could be observed at the base of ciliated cells in both nasal mucosa and bronchial epithelium (arrowheads). Rare ACE2 staining was present in a few alveolar cells (arrows). No staining was observed in nasal squamous epithelium, bronchioles, or submucosal glands in either tissue. Gender and age are shown for all individuals. Red and green colored squares mark the positions in the TMA cores shown as magnifications. Scale bar = 50µm. Scale bar for images in dashed squares = 10 µm. (re=respiratory; sq=squamous).


( B ) ACE2 protein abundance in different human tissues from ProteomicsDB , with the median expression presented in log10 ( iBAQ ) .

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(B) ACE2 protein abundance in different human tissues from ProteomicsDB, with the median expression presented in log10(iBAQ).


( C ) Western blot of ACE2 using five different human tissue lysates . For lung , two different lysates were used : one male ( left ) and one female ( right ) .

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(C) Western blot of ACE2 using five different human tissue lysates. For lung, two different lysates were used: one male (left) and one female (right).


( A ) Schematic overview of FMR1 , FXR1 , FXR2 , FMR2 and position of the 8 selected single nucleotide polymorphisms ( SNPs ) . Line represents introns , grey box at the beginning and end of each gene stands for UTR region and red boxes represent exons . Gene structure plots generated using FancyGene ( Rambaldi Ciccarelli , 2009 )

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(A) Schematic overview of FMR1, FXR1, FXR2, FMR2 and position of the 8 selected single nucleotide polymorphisms (SNPs). Line represents introns, grey box at the beginning and end of each gene stands for UTR region and red boxes represent exons. Gene structure plots generated using FancyGene (Rambaldi Ciccarelli, 2009)


( B ) Distribution of repeat polymorphism lengths in FMR1 of male GRAS schizophrenia patients and healthy controls . ( C ) Distribution of repeat polymorphism lengths in FMR2 of male GRAS schizophrenia patients and healthy controls .

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(B) Distribution of repeat polymorphism lengths in FMR1 of male GRAS schizophrenia patients and healthy controls. (C) Distribution of repeat polymorphism lengths in FMR2 of male GRAS schizophrenia patients and healthy controls.


Figure 2 : SNP overview and unbiased selection according to the standard operating procedure ( SOP ) developed for phenotype - based genetic association study ( PGAS ) approaches . ( A ) Genes from the ' broader fragile X family ' and their chromosomal position . ( B ) SNP numbers available through direct genotyping in the here used semi - custom genotyping array ( Affymetrix , Santa Clara , CA , USA ) . ( C ) SNPs fulfilling some of the first round of selection criteria ( ' functional ' = SNPs , i . e . located in promoter region , 3 ' UTR or coding sequence ; MAF = MAF ≥ 0 . 2 ; LD = SNPs that ' survive ' after linkage disequilibrium pruning : r² < 0 . 8 ) . Underlined are the 13 SNPs selected for the PGAS approach using the PAUSS ( selection requirements : fulfilled 2 of the above criteria or were functional ) . Not more than 3 SNPs per gene are selected to avoid overrepresentation of one gene . ( D ) SNPs with a tendency in PGAS ( see Fig 3A ) at single SNP basis went into the final accumulation model .

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Figure 2: SNP overview and unbiased selection according to the standard operating procedure (SOP) developed for phenotype-based genetic association study (PGAS) approaches. (A) Genes from the 'broader fragile X family' and their chromosomal position. (B) SNP numbers available through direct genotyping in the here used semi-custom genotyping array (Affymetrix, Santa Clara, CA, USA). (C) SNPs fulfilling some of the first round of selection criteria ('functional'=SNPs, i.e. located in promoter region, 3'UTR or coding sequence; MAF=MAF≥0.2; LD=SNPs that 'survive' after linkage disequilibrium pruning: r²<0.8). Underlined are the 13 SNPs selected for the PGAS approach using the PAUSS (selection requirements: fulfilled 2 of the above criteria or were functional). Not more than 3 SNPs per gene are selected to avoid overrepresentation of one gene. (D) SNPs with a tendency in PGAS (see Fig 3A) at single SNP basis went into the final accumulation model.


( A - B ) A total of 13 SNPs pre - selected according to the SOP presented in Fig 2 underwent PGAS screening as exemplified here : ( A ) PAUSS association pattern of an exemplary fictitious autosomal ( upper panel ) and a sex - chromosomal ( lower panel ) SNP which are eligible for the accumulation model . The genotype associated with the highest average PAUSS ( in this example CC ) is the ' proautistic genotype ' ( indicated by the black arrow ) and is assigned a score of 1 . The heterozygous genotype is assigned a score of 0 . 5 and the homozygous genotype associated with the lowest PAUSS receives a score of 0 . Please note that the difference between genotypes does not have to be statistically significant . ( B ) PAUSS association pattern of an exemplary fictitious autosomal ( upper panel ) and a sex - chromosomal ( lower panel ) SNP which would not be selected for the accumulation model because of unclear phenotypical / biological relevance .

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(A-B) A total of 13 SNPs pre-selected according to the SOP presented in Fig 2 underwent PGAS screening as exemplified here: (A) PAUSS association pattern of an exemplary fictitious autosomal (upper panel) and a sex-chromosomal (lower panel) SNP which are eligible for the accumulation model. The genotype associated with the highest average PAUSS (in this example CC) is the 'proautistic genotype' (indicated by the black arrow) and is assigned a score of 1. The heterozygous genotype is assigned a score of 0.5 and the homozygous genotype associated with the lowest PAUSS receives a score of 0. Please note that the difference between genotypes does not have to be statistically significant. (B) PAUSS association pattern of an exemplary fictitious autosomal (upper panel) and a sex-chromosomal (lower panel) SNP which would not be selected for the accumulation model because of unclear phenotypical/biological relevance.


( C - D ) The specificity of the association of the 8SNP accumulation model with an autistic phenotype ( as determined using PAUSS ; compare Fig 4B ) is controlled by applying an unrelated ( or ' non - sense ' ) phenotype , e . g . delusional - depression : ( C ) Intercorrelation pattern of single items included in the delusional - depression composite score , used here as example control phenotype . Cronbach ' s alpha is presented as measure of internal consistency . ( D ) The delusional - depression composite score is not associated with the number of proautistic genotypes of the 8SNP risk model in the discovery sample .

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(C-D) The specificity of the association of the 8SNP accumulation model with an autistic phenotype (as determined using PAUSS; compare Fig 4B) is controlled by applying an unrelated (or 'non-sense') phenotype, e.g. delusional-depression: (C) Intercorrelation pattern of single items included in the delusional-depression composite score, used here as example control phenotype. Cronbach's alpha is presented as measure of internal consistency. (D) The delusional-depression composite score is not associated with the number of proautistic genotypes of the 8SNP risk model in the discovery sample.


( A ) PAUSS ( PANSS autism severity score ) composition and item intercorrelation pattern in the GRAS sample of male schizophrenic individuals ( discovery sample ) . Cronbach ' s alpha is presented as measure of internal consistency and also provided for the male replication samples I and II .

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(A) PAUSS (PANSS autism severity score) composition and item intercorrelation pattern in the GRAS sample of male schizophrenic individuals (discovery sample). Cronbach's alpha is presented as measure of internal consistency and also provided for the male replication samples I and II.


( B ) Association of PAUSS with the number of proautistic genotypes of the 8SNP risk model in the discovery sample .

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(B) Association of PAUSS with the number of proautistic genotypes of the 8SNP risk model in the discovery sample.


( C ) PAUSS comparison of extreme groups with high and low numbers of accumulated proautistic genotypes in the discovery sample ; binary logistic regression analysis with non - z - standardized PAUSS as dependent variable ; mean ± SEM .

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(C) PAUSS comparison of extreme groups with high and low numbers of accumulated proautistic genotypes in the discovery sample; binary logistic regression analysis with non-z-standardized PAUSS as dependent variable; mean±SEM.


( D ) PAUSS comparison of extreme groups with high and low numbers of accumulated proautistic genotypes in replication sample I of male schizophrenia patients ; binary logistic regression analysis ; mean ± SEM .

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(D) PAUSS comparison of extreme groups with high and low numbers of accumulated proautistic genotypes in replication sample I of male schizophrenia patients; binary logistic regression analysis; mean±SEM.


( E ) PAUSS comparison of extreme groups with high and low numbers of accumulated proautistic genotypes in replication sample II of male disease control patients ; Mann - Whitney U test ; mean ± SEM .

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(E) PAUSS comparison of extreme groups with high and low numbers of accumulated proautistic genotypes in replication sample II of male disease control patients; Mann-Whitney U test; mean±SEM.


F ) The highly significant correlation of PAUSS and social support underlines the validity of social support as an autism proxy phenotype ; mean ± SEM .

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F) The highly significant correlation of PAUSS and social support underlines the validity of social support as an autism proxy phenotype; mean±SEM.


( G ) Comparison of the extreme groups with high and low numbers of accumulated proautistic genotypes for the autism proxy phenotype social support in the discovery sample ; binary logistic regression analysis ; mean ± SEM .

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(G) Comparison of the extreme groups with high and low numbers of accumulated proautistic genotypes for the autism proxy phenotype social support in the discovery sample; binary logistic regression analysis; mean±SEM.


( H ) Comparison of the extreme groups with high and low numbers of accumulated proautistic genotypes for the autism proxy phenotype social support in the male replication sample III from general population ; binary logistic regression analysis ; mean ± SEM . For all replications p - values for one - sided tests are shown .

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(H) Comparison of the extreme groups with high and low numbers of accumulated proautistic genotypes for the autism proxy phenotype social support in the male replication sample III from general population; binary logistic regression analysis; mean±SEM. For all replications p-values for one-sided tests are shown.


( A ) FXR1 expression in PBMC of individuals carrying the rs2601 GG ( low risk ; N = 16 ) versus AA genotype ( high risk ; N = 27 ) .

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(A) FXR1 expression in PBMC of individuals carrying the rs2601 GG (low risk; N=16) versus AA genotype (high risk; N=27).


( B ) PBMC microRNA expression was normalized and data is plotted from all 4 miR - 181 - 5p members ( miR - 181a , b , c , d - 5p ) .

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(B) PBMC microRNA expression was normalized and data is plotted from all 4 miR-181-5p members (miR-181a, b, c, d-5p).


( C ) Sequence homology of all 4 human mature miR - 181 species shown together with sequences in the broader fragile X gene family containing binding sites for the miR - 181 family ( seed matches identified using Target Scan Human and SFOLD ) . The red letters specify seed sequences and seed matches , respectively . Chromosome positions for each seed sequence and seed match are denoted ( human genome assembly GRCh38 / hg38 ) .

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(C) Sequence homology of all 4 human mature miR-181 species shown together with sequences in the broader fragile X gene family containing binding sites for the miR-181 family (seed matches identified using Target Scan Human and SFOLD). The red letters specify seed sequences and seed matches, respectively. Chromosome positions for each seed sequence and seed match are denoted (human genome assembly GRCh38/hg38).


( D ) Denoted are ΔΔG values for binding of each of the miR - 181 family members to different 3 ' UTR positions . Positions were identified using Target Scan Human and SFOLD and then processed using PITA algorithm to yield the denoted ΔΔG values . ΔΔG is an energetic score , and the more negative its value , the stronger is the expected binding of the microRNA to the given

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(D) Denoted are ΔΔG values for binding of each of the miR-181 family members to different 3'UTR positions. Positions were identified using Target Scan Human and SFOLD and then processed using PITA algorithm to yield the denoted ΔΔG values. ΔΔG is an energetic score, and the more negative its value, the stronger is the expected binding of the microRNA to the given site


( D ) The indicated amounts of recombinant wild - type and mutant PPM1H or PPM1J ( with a His - Sumo N - terminal tag , expressed in E . coli ) were incubated in vitro with 2 . 5 µg pThr72 phosphorylated Rab8a ( left ) or pThr73 phosphorylated Rab10 ( right ) for 20 min in the presence of 10 mM MgCl2 in 40 mM HEPES pH 7 . 5 buffer . Reactions were terminated by addition of SDS Sample Buffer and analyzed by Phos - tag gel electrophoresis that separates phosphorylated ( slow migrating ) and dephosphorylated Rabs . The gel was stained with Instant Blue Coomassie . D288A is a substrate - trapping ( inactive ) variant of PPM1H and was used as a control .

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(D) The indicated amounts of recombinant wild-type and mutant PPM1H or PPM1J (with a His-Sumo N-terminal tag, expressed in E. coli) were incubated in vitro with 2.5 µg pThr72 phosphorylated Rab8a (left) or pThr73 phosphorylated Rab10 (right) for 20 min in the presence of 10 mM MgCl2 in 40 mM HEPES pH 7.5 buffer. Reactions were terminated by addition of SDS Sample Buffer and analyzed by Phos-tag gel electrophoresis that separates phosphorylated (slow migrating) and dephosphorylated Rabs. The gel was stained with Instant Blue Coomassie. D288A is a substrate-trapping (inactive) variant of PPM1H and was used as a control.


( D ) Kinetics of phosphate hydrolysis . 25 nM recombinant wild - type PPM1H was incubated with increasing concentrations of pThr72 phosphorylated Rab8a ( GTPγS or GDP ) as described in Materials and Methods . Initial velocity ( V0 ) was calculated by dividing the concentration of released phosphatase ( μM ) by time ( min ) and plotted against substrate concentration for pThr72 phosphorylated Rab8a [ GTP bound conformation ] ( blue ) and pThr72 phosphorylated Rab8a [ GDP bound conformation ] ( red ) . The experiments were repeated twice , and both data points are shown in curves . Line fittings for the left and middle panel were performed using the mean of the two values . Kinetic constants ( Kcat , Vmax , Km ) were obtained using GraphPad software , and their uncertainties ( ± ) correspond to the SE of mean .

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(D) Kinetics of phosphate hydrolysis. 25 nM recombinant wild-type PPM1H was incubated with increasing concentrations of pThr72 phosphorylated Rab8a (GTPγS or GDP) as described in Materials and Methods. Initial velocity (V0) was calculated by dividing the concentration of released phosphatase (μM) by time (min) and plotted against substrate concentration for pThr72 phosphorylated Rab8a[GTP bound conformation] (blue) and pThr72 phosphorylated Rab8a[GDP bound conformation] (red). The experiments were repeated twice, and both data points are shown in curves. Line fittings for the left and middle panel were performed using the mean of the two values. Kinetic constants (Kcat, Vmax, Km) were obtained using GraphPad software, and their uncertainties (±) correspond to the SE of mean.