EMBO/SourceData huggingface dataset:
Figure 2A, B PBMC-derived macrophages were treated with GBS WT, ΔcylE, ΔcovR, or ΔcovRΔcylE at an MOI of 1 and incubated for 4 h. Cytotoxicity was measured by LDH release (A), and IL1β release in supernatants was measured by ELISA (B).Data information: Data shown are the average of four independent experiments performed in triplicate, error bars ± SEM. Significance was determined using Bonferroni's multiple comparison test following ANOVA. (A) n = 4, *P = 0.021 for WT versus ΔcovR; *P = 0.01 for ΔcovR versus ΔcovRΔcylE. (B) n = 4, *P = 0.031 for WT versus ΔcovR; *P = 0.036 for ΔcovR versus ΔcovRΔcylE. (C) n = 4, ****P < 0.0001, **P = 0.002. (D) n = 4, **P = 0.005.C, D PBMC-derived macrophages primed with 100 ng/ml LPS for 3 h were incubated with various concentrations of GBSpigment or control ΔcylE extract for 4 h. Cytotoxicity was measured by alamar blue assay (C), and IL1β release from pigment- or ΔcylE extract-treated cells was measured by Luminex assay (D). (C) n = 4, ****P < 0.0001, **P = 0.002. (D) n = 4, **P = 0.005.
Figure 3A, B WT THP-1macrophages were treated with GBS WT, ΔcylE, ΔcovR, or ΔcovRΔcylE at an MOI of 1 and incubated for 4 h. Cytotoxicity was measured by LDH release (A), and IL1β release in supernatants was measured by Luminex assay (B).Data information: Data from three independent experiments performed in triplicate are shown, error bars ± SEM. Significance was determined using Bonferroni's multiple comparison test following ANOVA. (A) n = 3, **P = 0.0080 for WT versus ΔcovR; **P = 0.0016 for ΔcovR versus ΔcovRΔcylE. (B) n = 3, *P = 0.03 for WT versus ΔcovR; *P = 0.02 for ΔcovR versus ΔcovRΔcylE. (C) n = 3, ****P < 0.0001. (D) n = 3, *P = 0.01.C, D WT THP-1macrophages were incubated with various concentrations of GBSpigment or control ΔcylE extract for 4 h. Cytotoxicity was measured by alamar blue assay (C), and IL1β release from pigment- or ΔcylE extract-treated cells was measured by Luminex assay (D).(C) n = 3, ****P < 0.0001. (D) n = 3, *P = 0.01.
Figure 4A, B THP-1macrophages transfected with empty vector, scrambled control, shASC, or shNLRP3 were treated with GBS WT, ΔcylE, ΔcovR, or ΔcovRΔcylE at an MOI of 1 and incubated for 4 h. Cytotoxicity was measured by LDH release (A), and IL1β release in supernatants was measured by Luminex assay (B). Data from three independent experiments performed in triplicate are shown.Data information: Data were analyzed using Bonferroni's multiple comparison test following ANOVA, error bars ± SEM. (A) n = 3, for WT: **P = 0.001 (vector versus shNLRP3), **P = 0.0016 (vector versus shASC); for ΔcovR: ***P = 0.0008 (vector versus shNLRP3), ***P = 0.0001 (vector versus shASC), *P = 0.047 (scrambled versus shNLRP3), **P = 0.0054 (scrambled versus shASC). Data obtained from THP-1/vector was not significantly different from THP-1/scrambled, P = 0.18. (B) n = 3, *P = 0.025. Data obtained from THP-1/vector were not significantly different from THP-1/scrambled, P = 0.89.C, D The shRNA THP-1macrophages were incubated with various concentrations of GBSpigment or ΔcylE extract for 4 h. Cytotoxicity was measured by alamar blue assay (C), and IL1β release in supernatants was measured by Luminex assay (D). Pigment-mediated cytotoxicity is dependent on the NLRP3 inflammasome components, suggesting that pigment is inducing a programmed cell death. Data shown are the average of at least three independent experiments performed in triplicate.Data information: Data were analyzed using Bonferroni's multiple comparison test following ANOVA, error bars ± SEM. (C) n = 3, for 4 μM pigment: **P = 0.0063 (vector versus shNLRP3), **P = 0.0035 (vector versus shASC), *P = 0.01 (scrambled versus shNLRP3), **P = 0.009 (scrambled versus shASC); for 2 μM pigment: **P = 0.0088 (vector versus shNLRP3), **P = 0.0066 (vector versus shASC), *P = 0.01 (scrambled versus shNLRP3 or shASC); for 1 μM pigment: *P = 0.02 (vector versus shNLRP3), *P = 0.01 (vector versus shASC). Data obtained from THP-1/vector were not significantly different from THP-1/scrambled, P = 0.99. (D) n = 3, ****P < 0.0001, *P = 0.015.
Figure 5A, B THP-1macrophages proficient for NLRP3 (transfected with scrambled control, A) or deficient for NLRP3 (shNLRP3, B) were treated with 1 μM pigment or ΔcylE extract for 20 min, and propidium iodide (PI) was added during the final 10 min. PI uptake was measured by flow cytometry, and data shown are representative of two independent experiments.C, D Intracellular potassium concentration was measured by ICP-AES. THP-1macrophages transfected with the scrambled control (C) or shNLRP3 (D) were treated with GBSpigment (1 μM) or an equivalent amount of the ΔcylE extract. At various time points, cells were lysed and intracellular [K+] was measured relative to untreated cells (see bars and left y-axis), and percent cell death was quantified by alamar blue (see squares, dotted connecting lines and right y-axis). Both NLRP3-proficient and NLRP3-deficient macrophages initially lose intracellular K+ due to GBSpigment (compare t = 0 min to t = 30 min), but the NLRP3-deficient cells (shNLRP3) are able to recover, while the scrambled control do not, demonstrating that initial K+ loss occurs independently of NLRP3. Data are average of three independent experiments performed with independent pigment preparations in triplicate and were analyzed using Dunnett's multiple comparison test following ANOVA; all data were compared to control at t = 0, error bars ± SEM. (C) n = 3, ***P = 0.0002, *P = 0.019, **P = 0.0032 for 120 min, **P = 0.0072 for 180 min. (D) n = 3, **P = 0.0043, *P = 0.031.(E) WT THP-1macrophages were incubated with pigment in media containing either 5 mM or 50 mM potassium chloride, and cytotoxicity was measured by alamar blue assay. The addition of potassium chloride is able to protect the macrophages from cytolysis, demonstrating that K+ efflux is essential for this process. Data shown are three independent experiments performed in triplicate and were analyzed using Bonferroni's multiple comparison test following ANOVA, error bars ± SEM (n = 3, ****P < 0.0001, **P = 0.0036).
Figure 6THP-1macrophages were treated with GBSpigment, and caspase 1 activation was measured by flow cytometry using a FLICA reagent. Pigment treatment of the scrambled shRNA control cell line induces more caspase 1 activation compared to the shNLRP3cell line, demonstrating that the pigment activates caspase 1 exclusively through the NLRP3 inflammasome. Results are representative of three independent experiments.WT THP-1macrophages were treated with the caspase 1 inhibitor Z-YVAD-FMK, the caspase 3/7 inhibitor Z-DEVD-FMK, or DMSO only prior to treatment with the GBSpigment. YVAD is able to significantly decrease cytotoxicity in cells treated with GBSpigment, demonstrating that caspase 1 is required for GBSpigment-mediated cell death, characteristic of pyroptosis, while DEVD had no effect. Data are average of three independent experiments, error bars ± SEM. Significance was determined using Bonferroni's multiple comparison test following ANOVA (n = 3, ****P < 0.0001, ***P = 0.0002, *P = 0.014).
Figure 7In utero fetal death in wild-type CD-1mice due to infection with GBS WT, hyperhemolytic ΔcovR, and non-hemolytic ΔcovRΔcylE. Fetal death is represented by the number of dead fetuses/total number of fetuses obtained from six pregnant mice per group. 'n' indicates total number of pups (both live and dead); ****P < 0.0001, Fisher's exact test.H&amp;amp;E staining of uterine tissue. Open arrow indicates the presence of few mononuclear cells, whereas filled arrows indicate increased infiltration of inflammatory cells and necrotic debris.Fetal death due to infection with hyperhemolytic GBS ΔcovR and non-hemolytic ΔcovRΔcylE in WT C57BL6 and NLRP3 inflammasome-deficient mice; fetal death is represented by the number of dead fetuses/total number of fetuses obtained from 6 pregnant mice per group. 'n' indicates total number of pups (both live and dead); *P = 0.011, **P = 0.0015, ****P < 0.0001, Fisher's exact test. Fetal death due to ΔcovRΔcylE in WT C57BL6 and NLRP3KO mice was not significant and is indicated as NS; P = 0.31, Fisher's exact test.Female pregnant wild-type (CD-1, C57BL6) or NLRP3-deficient mice were injected in utero with 106-7CFU of GBS WT, ΔcovR, or ΔcovRΔcylE and monitored for preterm birth. Surgery and GBS inoculation for each pregnant mouse were performed independently. Data shown are representative of experiments with 6 animals per group for each GBS strain and two animals were used for saline controls.Bacterial burden in fetal pups and uterine horns from mice infected with the various GBS strains (n = 6/pup; of note, pups that were delivered preterm were excluded from CFU enumeration. Scheme of pup numbering is shown in (A). RUH and LUH indicate right uterine horn and left uterine horn, respectively. CFUs are not significantly different between any of the groups (ANOVA, P = 0.6, error bars ± SEM).Female pregnant wild-type (CD-1, C57BL6) or NLRP3-deficient mice were injected in utero with 106-7CFU of GBS WT, ΔcovR, or ΔcovRΔcylE and monitored for preterm birth. Surgery and GBS inoculation for each pregnant mouse were performed independently. Data shown are representative of experiments with 6 animals per group for each GBS strain and two animals were used for saline controls.IL-1β levels in GBS-infected tissues (placenta and fetus, n = 6/group) was measured by Luminex assay (*P = 0.025, ***P = 0.0002, ****P < 0.0001, Bonferroni's multiple comparison test following ANOVA). IL-1β levels was not significantly different in NLRP3KO mice infected with ΔcovR compared to NLRP3KO mice infected with ΔcovRΔcylEmice and is indicated as NS; P = 0.99.
f1(d) NIH3T3 cells stably expressing PS-CFP2 proteins were exposed to a 405 nm light for the indicated times and photoconverting was monitored by imaging cells in the green and blue channels.(e, f) Mouse fibroblasts (NIH3T3) stably expressing PS-CFP2 or KFERQ-PS-CFP2 were photoconverted and maintained in media supplemented (+) or not (−) with serum for 16 h. (e) Representative images in both channels. (f) Quantification of the number of green fluorescent puncta per cell in cells maintained in the presence (white bars) or absence (gray bars) of serum. Values are mean+s.e. of four different experiments with >50 cells counted per experiment. (*) Differences with PS-CFP2 in the green channel (P=0.005, ANOVA-Bonferroni). (§) Differences with samples in the presence of serum (P=0.03, ANOVA-Bonferroni). (g) Green fluorescence intensity at excitation 450 nm and emission 510-550 nm. Fluorescence intensity in each sample was normalized by the number of cells present in the sample and the percentage of intensity at time 0 still remaining after 16 h of incubation in the presence (white bars) or absence (gray bars) of serum was calculated for each condition. Values are expressed relative to the decay observed in cells maintained in the presence of serum and are means+s.e. of 3 independent experiments. Differences with serum (*) were significant for P=0.04 (t-test). Scale bars, 5 μm.
f2(a-c) Temporal changes in the green fluorescence pattern of photoactivated NIH3T3 cells stably expressing KFERQ-PS-CFP2 maintained in the absence (black circles) or presence (white squares) of serum for the indicated times. (a) Representative images of cells maintained in serum-free media. Quantification of the number of puncta per cell (b) and green fluorescence intensity (c). Values are expressed as percentage of fluorescence at time 0 and half-lives (t1/2) are indicated. Differences in the number of puncta and fluorescence intensity between cells maintained in the presence and absence of serum (*) were significant for P=0.048 (16 h) and P=0.04 (24 h) (t-test).(d) The same cells imaged in the cyan channel, before and at the indicated times after photoconversion (arrow) by exposure to 405 nm light. Top: Representative images of the cyan fluorescence. Bottom: Quantification of the intensity of the cyan fluorescence. Values are expressed as times the intensity before photoconversion that was given an arbitrary value of 1. Values are mean+s.e. of 3 different experiments with >10 cells counted per experiment. Scale bars, 5 μm.
f3(a) Localization of photoconverted KFERQ-PS-CFP2 (green) in LysoTracker-stained compartments (red) in mouse fibroblasts maintained in the presence or absence of serum.(b) Percentage of co-localization of KFERQ-PS-CFP2 with the indicated proteins. Values are mean+s.e. of three different experiments with >50 cells counted per experiment.(c) Co-localization of photoconverted KFERQ-PS-CFP2 (green) with LAMP-1, LAMP-2 or LAMP-2A.(d) Immunofluorescence with antibodies that recognize PS-CFP2 (green) and hsc70 (red) in cells maintained in the absence of serum and fixed with methanol to eliminate the soluble cytosolic fraction.(e, f) Co-localization of photoconverted KFERQ-PS-CFP2 (green) with LC3, CD-M6PR, Rab5 (e) and ubiquitin (f) in mouse fibroblasts maintained in serum-free media. Scale bars, 5 μm.
f4(a) Mouse fibroblasts knockeddown for LAMP-2A and transfected with KFERQ-PS-CFP2 were photoconverted and maintained in media supplemented (+) or not (−) with serum for 16 h. Representative images of cells also immunostained for LAMP-1 are shown.(b, c) Mouse fibroblasts stably expressing KFERQ-PS-CFP2 were photoconverted and maintained for 16 h in media supplemented (+) or not (−) with serum in the presence of 3-methyladenine (3-MA) to inhibit macroautophagy. (b) Representative images of the 3-MA-treated cells. (c) Quantification of the number of green fluorescence puncta per cell in 3-MA treated or not cells maintained in the presence (white bars) or absence (black bars) of serum. Values are mean+s.e. of 3 different experiments with >50 cells counted per experiment. No significant differences were detectable between samples supplemented or not with 3MA. Scale bars, 5 μm.
f5(a) Immunoblot with the antibody that recognizes PS-CFP2 of homogenates (Hom), cytosol (Cyt) and lysosomal membranes (MB) and matrices (Mtx) isolated from mouse fibroblasts stably expressing PS-CFP2 or KFERQ-PS-CFP2 proteins 16 h after serum removal.(b) Immunoblot for the indicated proteins of homogenates (Hom), mitochondria (Mit) and lysosomal membranes and matrices isolated from cells stably expressing KFERQ-PS-CFP2 maintained for 16 h in media supplemented (+) or not (−) with serum. (c) Quantification of the amount of KFERQ-PS-CFP2 associated to lysosomes (matrix and membrane) performed in blots as the one shown in (b). Values are expressed as percentage of the total KFERQ-PS-CFP2 in the cell and are mean+s.e. of three different experiments. Difference in the lysosomal associated KFERQ-PS-CFP2 between serum+ and serum− condition (*) were significant for P=0.033 (t-test).(d) Changes in the intracellular content of KFERQ-PS-CFP2 on serum removal. Values are expressed as percentage of KFERQ-PS-CFP2 present in serum-supplemented cells and area mean+s.e. of three different experiments. Values are mean+s.e. of three different experiments and differences are significant (*) for P=0.007 (t-test).(e-g) Effect of inhibition of macroautophagy by treatment with 3-methyladenine (3-MA) on the lysosomal content of KFERQ-PS-CFP2 in cells maintained in serum-free media for 16 h. (e) Immunoblot for the indicated proteins of the same fractions as described in (b). The amount of KFERQ-PS-CFP2 associated to lysosomes (f) and the changes in the intracellular content of KFERQ-PS-CFP2 were calculated as in (c, d). Values are mean+s.e. of three different experiments and differences are significant (*) for P=0.032 (t-test).
f6(a, b) Human neuroblastoma (SH-SY5Y), hepatoma (HuH-7) and breast adenocarcinoma (MCF-7) cell lines were transiently transfected with KFERQ-PS-CFP2, photoconverted and maintained in media supplemented (+) or not (−) with serum for 16 h. Images show representative cells after immunostaining for LAMP-2 (red). (b) Quantification of the number of green fluorescent puncta per cell under basal conditions (black bars) and after serum removal (gray bars). Values of NIH3T3 are included as reference. Values are mean+s.e. of three-to-five different experiments with >50 cells counted per experiment. Differences with (*) NIH3T3 or (§) with cell supplemented with serum are significant for P0.05 (range 0.002-0.03, ANOVA-Bonferroni).(c) Primary mouse skin fibroblasts were transduced with a lentiviral vector for expression of KFERQ-PS-CFP2, photoconverted and maintained in media supplemented (+) or not (−) with serum for 16 h. Left: representative image in the green channel. Right: Quantification of the number of green fluorescent puncta per cell in cells maintained in the presence (black bars) or absence (gray bars) of serum. Values are mean+s.e. of three different experiments with >50 cells counted per experiment. (P=0.002, ANOVA-Bonferroni).(d) Mouse fibroblasts stably expressing KFERQ-PS-CFP2 were plated in 384-well plates, maintained in the presence (white bar) or absence (black bars) of serum and subjected to treatment with 3-methyladenine (3-MA), the proteasome inhibitor MG-132 or H2O2. In both (d, e), after fixation, images were subjected to high content image analysis and the percent of responders was calculated in >200 cells per condition. Values are mean+s.e. of results in three different experiments. *P0.001 (range 0.0002-0.0007, t-test). Scale bars, 5 μm.(e) Mouse fibroblasts stably expressing KFERQ-PS-CFP2 were plated in 384-well plates, maintained in the presence of serum supplemented media and leave untreated (white bar) or subjected to the indicated treatments (black bars). In both (d, e), after fixation, images were subjected to high content image analysis and the percent of responders was calculated in >200 cells per condition. Values are mean+s.e. of results in three different experiments. *P0.001 (range 0.0002-0.0007, t-test). Scale bars, 5 μm.
f7(b) Mouse fibroblasts (NIH3T3) stably expressing KFERQ-PA-mCherry1 were photoactivated and maintained in media supplemented (+) or not (−) with serum for 16 h. Left: Representative images. Nuclei are staining with DAPI. Right: Quantification of the number of red fluorescent puncta per cell. Values are mean+s.e. of three different experiments with >20 cells counted per experiment. *P=0.001, ANOVA-Bonferroni.(c) Temporal changes in the mCherry fluorescent intensity in cells stably expressing KFERQ-PA-mCherry1 at the indicated times after photoactivation using FACS analysis. Cells were maintained in media supplemented (white squares) or not (black circles) with serum, and fluorescence by the MFI was calculated on events gated on living cells. Values are expressed as percentage of the intensity at time 0 and are mean+s.e. of 3-4 individual experiments.(d) Representative FACS experiment showing the effect of serum removal at 6 h (top) and 42 h (bottom). PS, photoswitching. Scale bars, 5 μm.
Figure 1Mouse qPCR array analysis was performed with Colo357 and MiaPaca-2 orthotopic tumor samples treated with saline (control) or 2G8 (n = 3/group) to detect changes of mouse stromal gene expression after treatment. A heat map was generated with ratios of gene expression (2G8 vs. control).ELISA-based Quansys mouse chemokine assay was performed with Capan-1 (Cap), MiaPaCa-2 (Mia), Colo357 (Colo), and C5LM2 (C5) orthotopic tumor samples treated with saline (control) or 2G8 (n = 3/group) to detect mouse chemokine changes after treatment. Change of mouse IL-6 was shown.KIC were treated for 4 weeks and KPC mice were treated for 55 days with Mac84 (control) or 2G8. Tumors from were collected for mouse IL-6 ELISA (n = 6/group).KIC mice were treated for 4 weeks and KPC mice were treated for 55 days with Mac84 (control) or 2G8. The activation of SMAD2 (P-Ser465/467) (E and H-I), STAT3 (P-Tyr705) (F and J-K) and expression of IL-6R (G) was detected by immunohistochemistry (n = 4/group).
Figure 2Single-cell RNA sequencing was performed to profile cell populations in normal mouse pancreas (n = 2), early KIC (40-day-old, n = 2) and late KIC (60-day-old, n = 3) pancreata. Samples from the same stage were pooled. Violin plots of expression of Il6, Tgfbr1 and Tgfbr2 in distinct cell populations is shown.Expression of TGFβR1 and TGFβR2 in cell lysates harvested from KIC (mPLRB8, mPLRB9), KPC (KPC-M01, KPC-M09) and KPfC (BMFA3, CT1BA5) mouse cancer cells, mouse macrophages (RAW 264.7), and mouse fibroblasts (NIH 3T3 and pancreatic stellate cells). RAW 264.7 cells were induced into M1 (30 ng/ml LPS for 18 hours) or M2 (20 ng/ml IL-4 for 18 hours) macrophages. Tubulin was used as a loading control.Pearson and Spearman correlation of the expression of IL6 and TGFBR2 in PDA patients from TCGA (n = 223) using R.
Figure 3Control, M1 (LPS stimulated) and M2 (IL-4 stimulated) RAW 264.7 cells, as well as NIH 3T3 cells were treated with TGFβ (30 ng/ml), or TGFβ + 2G8 (100 ng/ml) for 24 hours. Cell lysates were harvested and blotted for P-SMAD2 (P-Ser465/467), SMAD2, and tubulin (A).Control, M1 (LPS stimulated) and M2 (IL-4 stimulated) RAW 264.7 cells, as well as NIH 3T3 cells were treated with TGFβ (30 ng/ml), or TGFβ + 2G8 (100 ng/ml) for 24 hours. Conditioned media (CM) was collected mouse IL-6 ELISA (B-C).NIH 3T3 (D), pancreatic stellate cells (PSC) (E) and human CAF cell lines CAF-PC1 and CAF-PC2 (F) were treated with TGFβ (30 ng/ml) and/or IL-1α (1 ng/ml) for 24 hours. CM was collected for mouse or human IL-6 ELISA.CAF-PC2 cells were treated with TGFβ (30 ng/ml) for 24 hours with siRNA-mediated knockdown of JUNB, JUND, SMAD4 or RELA. CM was collected and subjected to human IL-6 ELISA.CAF-PC2 cells were treated with IL-1α (1 ng/ml) (H) for 24 hours with siRNA-mediated knockdown of JUNB, JUND, SMAD4 or RELA. M was collected and subjected to human IL-6 ELISA.KIC (mPLRB9), KPC (KPC-M09) and KPfC (BMFA3) cell lines were treated with normal DMEM (CTRL), CM from NIH 3T3 (CM), CM from TGFβ-treated NIH 3T3 (TGFβ-CM), CM from TGFβ-treated NIH 3T3 + 2G8 (TGFβ-CM + 2G8) normal DMEM + TGFβ (TGFβ), CM from TGFβ-treated NIH 3T3 + IL-6 neutralizing antibody (TGFβ-CM + IL-6 Ab). Cell lysates were harvested and blotted for P-STAT3 (P-Tyr705), STAT3, P-SMAD2 (P-Ser465/467), SMAD2, and tubulin (J).3D culture: cells were seeded on poly-HEMA-coated 96-well plates and cultured for 4 days (5000 cancer cells for monoculture, 3000 cancer cells + 2000 NIH 3T3 for co-culture). IL-6 neutralizing antibody (100 ng/ml).3D culture: cells were seeded on poly-HEMA-coated 96-well plates and cultured for 4 days (5000 cancer cells for monoculture, 3000 cancer cells + 2000 NIH 3T3 for co-culture). IL-6 neutralizing antibody (100 ng/ml).